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Master Thesis
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1 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science CHEMICAL COMPOSITION OF FLORAL VOLATILES AND EXPRESSION OF SCENT-RELATED GENES IN VANDA MIMI PALMER By MOHD HAIRUL AB. RAHIM November 2010 Chairman: Parameswari a/p Namasivayam, PhD Faculty: Biotechnology and Biomolecular Sciences Vanda Mimi Palmer is an orchid hybrid of Vanda Tan Chay Yan and Vanda tessellata. The flower of this orchid produces a sweet fragrance during daylight hours at the open- flower stage. Lately, a lot of effort has been channeled into understanding the fragrance pathway in scented flowers but none in Vandaceous orchids. This study aims to investigate on the molecular and biochemical aspects of the fragrance in Vanda Mimi Palmer. Scent emission analysis of this orchid was carried out at different developmental stages and at different time points in a 24-hour cycle. Gas chromatography mass spectrometry (GC-MS) analysis has shown that the scent of Vanda Mimi Palmer is dominated by metabolites from the terpenoid, benzenoid and phenylpropanoid pathways. Identified volatile compounds derived from terpenoid pathway are linalool, ocimene and nerolidol. Meanwhile, methylbenzoate, phenylethanol, benzyl acetate and phenylethyl acetate are the metabolites identified from the benzenoid and phenylpropanoid pathways. Scent emission of Vanda Mimi Palmer is also developmentally and temporally regulated.
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Page 1: Vanda Mimi Palmer Thesis

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of

the requirement for the degree of Master of Science

CHEMICAL COMPOSITION OF FLORAL VOLATILES AND EXPRESSION

OF SCENT-RELATED GENES IN VANDA MIMI PALMER

By

MOHD HAIRUL AB. RAHIM

November 2010

Chairman: Parameswari a/p Namasivayam, PhD

Faculty: Biotechnology and Biomolecular Sciences

Vanda Mimi Palmer is an orchid hybrid of Vanda Tan Chay Yan and Vanda tessellata.

The flower of this orchid produces a sweet fragrance during daylight hours at the open-

flower stage. Lately, a lot of effort has been channeled into understanding the fragrance

pathway in scented flowers but none in Vandaceous orchids. This study aims to

investigate on the molecular and biochemical aspects of the fragrance in Vanda Mimi

Palmer. Scent emission analysis of this orchid was carried out at different developmental

stages and at different time points in a 24-hour cycle. Gas chromatography mass

spectrometry (GC-MS) analysis has shown that the scent of Vanda Mimi Palmer is

dominated by metabolites from the terpenoid, benzenoid and phenylpropanoid pathways.

Identified volatile compounds derived from terpenoid pathway are linalool, ocimene and

nerolidol. Meanwhile, methylbenzoate, phenylethanol, benzyl acetate and phenylethyl

acetate are the metabolites identified from the benzenoid and phenylpropanoid pathways.

Scent emission of Vanda Mimi Palmer is also developmentally and temporally regulated.

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On the molecular biology aspect, fragrance-related cDNA transcripts have been identified

by a differential screening of the Vanda Mimi Palmer‟s floral cDNA library. Reverse-

Northern analysis was carried out by hybridizing the putative positive clones with two

cDNA probes representing mRNA transcripts of bud and fully-open flower during the

daylight hour separately. The clones that showed up-regulated expression in fully-open

flowers were selected for sequencing. From the sequencing results, putative 4-(cytidine

5′-diphospho)-2-C-methyl-D-erythritol kinase (VMPCMEK), putative cytochrome P450

(VMPCyP450), and an unknown protein (VMPA28) were selected for molecular

characterization. The three transcripts with a putative phenylacetaldehyde synthase

(VMPPAAS), a previously isolated transcript from Expressed Sequence-Tags (ESTs),

were subjected to full-length cDNA isolation and expression analyses by real-time RT-

PCR. Expression analyses of these transcripts were investigated in different tissues, at

different developmental stages, and time points in a 24-hour cycle using real-time RT-

PCR. The transcripts are highly expressed in floral tissues compared to vegetative tissues

as well as developmentally and temporally regulated. In conclusion, from the

biochemical and molecular work on the fragrance, there are two putative biochemical

pathways which might be involved in fragrance biosynthesis in Vanda Mimi Palmer that

are the terpenoid, and also the benzenoid and phenylpropanoid pathways.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

KOMPOSISI KIMIA HARUMAN DAN EKSPRESI GEN-GEN BERKAITAN

WANGIAN DALAM VANDA MIMI PALMER

Oleh

MOHD HAIRUL AB. RAHIM

November 2010

Pengerusi: Parameswari a/p Namasivayam, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Vanda Mimi Palmer ialah orkid kacukan di antara Vanda Tan Chay Yan dan Vanda

tesellata. Bunga orkid ini yang telah berkembang sepenuhnya mengeluarkan bau yang

harum pada waktu siang. Kebelakangan ini, perhatian diberikan terhadap tapak jalan

biokimia penghasilan wangian bagi bunga wangi selain daripada orkid Vanda. Kajian ini

bertujuan untuk mengkaji wangian Vanda Mimi Palmer yang merangkumi aspek-aspek

biokimia dan biologi molekul. Analisis bagi wangian yang dihasilkan oleh orkid ini

dijalankan pada setiap peringkat perkembangan bunga dan juga masa yang berbeza dalam

kitaran 24 jam sehari. Analisis dijalankan menggunakan alat kromatografi gas-

spectrometrik jisim (GC-MS). Analisis GC-MS tersebut menunjukkan wangian Vanda

Mimi Palmer didominasi oleh metabolit daripada tapak jalan terpenoid, dan juga,

benzenoid dan phenylpropanoid. Pengeluaran wangian Vanda Mimi Palmer juga didapati

dikawalatur mengikut peringkat perkembangan bunga dan juga peredaran masa. Dalam

aspek biologi molekul, transkrip cDNA berkaitan penghasilan wangian telah dikenalpasti

melalui penyaringan pembezaan perpustakaan cDNA bunga (floral cDNA library) Vanda

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Mimi Palmer. Analysis „reverse-Northern‟ dijalankan dengan menghibridkan secara

berasingan klon-klon positif bersama dua „cDNA probe‟ yang berbeza mewakili transkrip

mRNA bagi peringkat kudup dan juga bunga kembang penuh. Klon-klon yang

menunjukkan ekspresi yang lebih tinggi bagi peringkat bunga kembang penuh

berbanding kudup dipilih untuk jujukan. Daripada keputusan analisis jujukan, 4-(cytidine

5′-diphospho)-2-C-methyl-D-erythritol kinase putatif (VMPCMEK), cytochrome P450

protein putatif (VMPCyP450), dan transkrip protein yang belum dikenalpasti (VMPA28)

dipilih untuk pencirian biologi molekul. Ketiga-tiga transkrip tersebut bersama transkrip

phenylacetaldehyde synthase putatif (VMPPAAS) yang dipencilkan daripada “Expressed

Sequence-Tags” (ESTs) bunga Vanda Mimi Palmer dipilih untuk pencirian termasuk

pemencilan cDNA lengkap dan juga analisis ekspresi menggunakan tindakbalas rantaian

polimerase masa nyata (RT-PCR). Analisis ekspresi tersebut dijalankan bagi tisu yang

berbeza, peringkat perkembangan bunga yang berbeza dan masa yang berbeza dalam

kitaran 24 jam sehari menggunakan RT-PCR. Transkrip tersebut menunjukkan ekspresi

yang tinggi pada tisu bunga berbanding tisu vegetatif, dan dikawalatur oleh peringkat

perkembangan bunga dan juga peredaran masa. Kesimpulannya, daripada hasil kajian

biokimia dan biologi molekul, dua tapak jalan biokimia telah dikenalpasti

berkemungkinan terlibat bagi penghasilan wangian dalam Vanda Mimi Palmer iaitu tapak

jalan terpenoid dan juga tapak jalan benzenoid dan phenylpropanoid.

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ACKNOWLEDGEMENTS

I would like to express my utmost gratitude to my supervisor, Dr. Parameswari a/p

Namasivayam, for her patience, encouragement, time as well as precious advice and

guidance, leading me throughout this research project. My sincere appreciation is also

extended to my co-supervisors, Assoc. Prof. Dr. Janna Ong Abdullah and Prof. Dr.

Gwendoline Ee Cheng Lian for their guidance, support and technical advice.

Special thanks to Malaysia Toray Science Foundation (MTSF) for giving me a research

grant for the screening and isolation of putative fragrance-related cDNAs. Another

special thanks to Universiti Putra Malaysia for supporting my work on biochemical and

molecular characterization of the fragrance of Vanda Mimi Palmer through Research

University Grant Scheme (RUGS) and also for providing me my stipend for the last two

years through Graduate Research Fellowship (GRF). My deepest appreciation also goes

to Chemistry Department, Faculty of Science UPM for giving me permission to use GC-

MS for my biochemical analysis of the scent of Vanda Mimi Palmer and also to En.

Zainal Abidin Kasim for his help in my biochemical analysis with GC-MS.

I would like to thank all my lab mates in Molecular Biology Laboratory, Biotech 3, UPM

as well as all of the laboratory staff in the laboratory, for their technical guidance,

support, care, forgiveness, valuable ideas and experience. My deepest appreciation is

extended to family for their endless support, care and love, accompanying me through all

the happiness and sadness in my study.

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CHAPTER 1

INTRODUCTION

Floral scent or floral fragrance is an important constituent for perfume and food

industries. The extracts from flowers including jasmine and rose have been used

extensively in fragrance and flavour industries. Besides that, there is always a high

demand for scented flowers from aromatherapy industries especially in Malaysia and

Thailand. In horticultural and agricultural industries, floral scent is very important for

pollination of crops. Floral scent studies have been well established in some scented

flowers including Clarkia breweri, Antirrhinum majus, Rosa hybrida and Petunia

hybrida in both biochemical and molecular aspects covering three fragrance biosynthetic

pathways that are terpenoid, lipoxygenase-catalyzed fatty acid derivatives, and also

benzenoid and phenylpropanoid pathways (Pichersky and Dudareva, 2007).

Orchids with fragrance have higher demand in the orchid industry and fetching higher

prices compared to non-fragrance orchids (Eric Kok, Manager, Malaysian Orchids Sdn.

Bhd., pers. comm. on 20th

May 2008). In orchid industry, extensive work has been

focused on hybridizing scented orchid with non-scented orchid in order to produce flower

with attractive colour appearances. Most of the progenies produced have diluted scent or

no scent at all. In orchids, floral scent identification started in the early 1990s (Kaiser,

1993) but the knowledge on fragrance biosynthetic pathways is still far from being

understood. More recently, a few fragrance-related cDNAs were identified from

expressed sequence-tags (ESTs) of Phalaenopsis bellina (Hsiao et al., 2006) and the only

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cDNA that has been well characterized is geranyldiphosphate synthase that is involved in

the biosynthesis of geranyl diphosphate, a precursor for monoterpenes biosynthesis

(Hsiao et al., 2008).

Besides Phalaenopsis bellina there are a lot of fragrance orchids which are still not well

studied for their fragrance characteristics including Vanda Mimi Palmer. Vanda Mimi

Palmer is a well-known commercial orchid hybrid especially in Malaysia and Thailand.

This orchid produces a sweet smelling fragrance during day time in fully-open flower

stage (Janna et al., 2005). This orchid had won several awards for its sweet smelling

fragrance including the Champion Award for Fragrant Orchid organized by the Royal

Horticultural Society of Thailand in 1993 and the Best Orchid Fragrance in the 17th

World Orchid Conference in 2002 (Nair and Arditti, 2002). Thus, Vanda Mimi Palmer

with its fragrance emission characteristic was chosen for this study in order to understand

its fragrance biosynthetic pathways.

The knowledge on the sequences of fragrance-related cDNAs isolated from Vanda Mimi

Palmer can be used for transformation into non-scented orchids and other non-scented

flowers in order to increase the commercial value of the orchids and other ornamental

flowers. Besides that, understanding on the fragrance biosynthetic pathways of Vanda

Mimi Palmer will assist in the cloning of fragrance-related cDNAs into bacterial and

yeast expression vector for production of fragrance compounds in bulk. The knowledge

of the proportion of each compound in the fragrance of Vanda Mimi Palmer will facilitate

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the production of custom-made perfume of the same smell as Vanda Mimi Palmer either

biologically or chemically synthesized.

The specific objectives for this study were:

1) to determine the constituents of the scent of Vanda Mimi Palmer in comparison to

its parents,

2) to isolate and characterize selected putative fragrance-related transcripts of Vanda

Mimi Palmer, and

3) to analyze the expression profile of the selected putative fragrance-related cDNAs

of Vanda Mimi Palmer

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CHAPTER 2

LITERATURE REVIEW

2.1 Orchid – An Introduction

Orchids are classified under the Orchidaceae, one of the largest families of flowering

plants with an estimated population of 20,000 to 35,000 species (Dressler, 1993;

Mabberly, 1997). More than 800 orchid genera have been identified from the entire world

including Aranda, Aranthera, Cattleya, Dendrobium, Oncidium, Phalaenopsis,

Paphiopedilum and Vanda. In Malaysia, there are more than 120 genera and 2000 species

that have been discovered (Hamdan, 2008). An orchid flower consists of three sepals and

three petals. The petals and sepals are usually nearly alike where petals are located in the

first whorl while sepals in the second whorl of the flower. One of the petals is often

highly modified to form the lip or labellum, and is complicated in shape (Seidenfaden

and Wood, 1992).

The habitats of orchids vary such as mountainous forests, highlands, tropical mountain

forests and also lowlands (Fadelah et al., 2001). In nature, there are epiphytic orchids

which grow on branches and trunks of trees, terrestrial orchids which grow on soil and

lithophyte orchids which grow on rocks (Hamdan, 2008). The epiphytic orchids use

branches and trunks of trees only for support purpose without taking anything from the

trees. Living up on the trees helps the orchids to get away from competition with other

plants on the forest floor and escape from mineral contaminants on soil (Rittershausen

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and Rittershausen, 2008). The source of nutrients for epiphyte and lithophyte are from

organic substances of dead leaves, mosses and insects meanwhile for terrestrial orchids,

the nutrients for growth are directly from the soil (Hamdan, 2008).

To date, more than 100,000 orchid hybrids have been established in the world either by

crossing between the same genera (interspecific hybrid) or by crossing with different

genera (intergeneric hybrid) (Hands, 2006). The first orchid hybrid in the world is

Calanthe Dominiyi produced in 1856, a cross of Calanthe masuca and Chalanthe furcata

(Sheela, 2008). The list of new orchid hybrids is now controlled by The Royal

Horticultural Society, England (RHS). Lately, a few hundreds orchid hybrids with

commercial values are established annually for orchid industry (Hamdan, 2008). In

Malaysia, the Vandaceous hybrids like Dendrobiums and Oncidiums are the most popular

cut-flower cultivated since they are easily grown and cultivated in Malaysia‟s climate

(Fadelah et al., 2001).

In the floriculture industry, the demand on orchid species and orchid hybrids is very high

from all over the world. The demand on orchids is high due to their aesthetic values

(exotic and limited sources) including colour, scent appearance and morphology. In

orchid industry, the price of scented orchids is often higher compared to scentless orchids

(Eric Kok, Manager, Malaysian Orchids Sdn. Bhd., pers. comm. on 20th

May 2008).

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2.2 Vanda Mimi Palmer and Its Parents

Vanda Mimi Palmer is a cross between Vanda tessellata and Vanda Tan Chay Yan

(Motes, 1997) (Figure 1). The special characteristic of Vanda Mimi Palmer compared to

other orchid hybrids is the fragrance characteristic. Vanda Mimi Palmer has won a few

international awards for its strong sweet fragrance such as the Champion Award for

Fragrant Orchid organized by the Royal Horticultural Society of Thailand in 1993 and the

Best Orchid Fragrance in the 17th

World Orchid Conference in 2002 (Nair and Arditti,

2002).

Vanda Mimi Palmer could have inherited its fragrance and colour characteristics from

Vanda tessellata, an epiphytic orchid from Sri Lanka, India and Burma (Kaiser, 1993;

Motes, 1997). Vanda tessellata has inflorescences of 25cm to 30cm long and grey-green-

brown flowers. The lip is white at the side and violet purple in the middle. The shape of

Vanda Mimi Palmer‟s flower closely resembles Vanda Tan Chay Yan‟s which is a hybrid

of Dutch Vanda Josephine and Vanda dearei (Yeoh, 1978). Vanda Tan Chay Yan has

round and flat petals and sepals. This hybrid has won many awards such as First Class

certificate (the highest award of the Royal Horticultural Society) in 1954, the Trophy for

The Best Vanda at the Second World Conference in Hawaii and numerous Singapore and

Malayan Prizes. In 1960s, this hybrid lost its popularity as commercial cut orchid due to

its disability to flower more than twice a year (Yeoh, 1978).

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Vanda tessellata Vanda Tan Chay Yan

Vanda Mimi Palmer

Figure 1: Vanda Mimi Palmer and Its Parents. Vanda Mimi Palmer is a hybrid of

Vanda tessellata (adapted from Hamdan, 2008) and Vanda Tan Chay Yan.

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In Malaysia, the demand for Vanda Mimi Palmer is high due to the fragrance emitted by

its flower rather than its beautiful colour and structure (Eric Kok, Manager, Malaysian

Orchids Sdn. Bhd., pers. comm. on 20th May 2008). Floral extracts from Vanda tessellata

(one of the parents of Vanda Mimi Palmer) have been used in some local traditional

practices for medicinal purposes in India, such as treatment for inflammatory conditions

and instilled into the ear as remedy for otitis (Chopra et al., 1956). Besides that, the

extract from the leaves in the form of paste is applied to the human body for cooling

down a fever (Chopra et al., 1956; Basu et al., 1971). Root extract from Vanda tessellata

had also been used for rheumatism treatment, fever, dyspepsia, bronchitis and also

nervous problem (Kirtikar and Basu, 1975). A scientific study on the extracts of Vanda

tessellata has shown inflammatory property against acute inflammation induced by

carrageenan, serotonin and formaldehyde (Suresh Kumar et al., 2000). Besides that,

alcohol extract from Vanda tessellata has shown an enhancement of male sexual activity

in normal mice (Suresh Kumar et al., 2000). Thus, Vanda Mimi Palmer might have some

medicinal properties as Vanda tessellata since half of the gene pool of Vanda Mimi

Palmer was derived from Vanda tessellata.

2.3 The Biological Importance of Floral Scent

In general, floral buds do not have scent, and the fragrance characteristic of a flower

appears during anthesis as the petals open (Schade et al., 2001). Floral scent emission

patterns vary among species. Some flowering plants such as Citrus medica and

Odontoglossum constrictum emit scent primarily during day time meanwhile some other

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plants such as Petunia hybrida and Clarkia breweri emit their scent at the highest level

during night time (Altenburger and Matile, 1988). Floral scent emission patterns are

different among species due to the control of cicardian clock, photoperiod and also

adaptation to specific pollinators‟ active time (Verdonk et al., 2003).

Floral scent is one of the factors that attract pollinators to help in pollination. The

pollinator varies among plant species including birds, insects and bats. Some flowering

plants need very specific pollinators for their pollination (Dobson, 1994) as they are

attracted to specific odors or scent emitted by flowering plants. For example, beetles are

attracted to flowers that have musty, spicy and fruity odors (Kaiser, 1993; Frowine, 2005)

while bees and flies are attracted and help in pollination of sweet scented flowers that can

be detected by human nose (Reinhard et al., 2004).

Besides pollination purpose, some plants emit volatiles such as monoterpenes,

sesquiterpenes and hormones such as salicylic acid, jasmonic acid and ethylene from

their vegetative tissues to defend themselves against pathogenic microorganisms and

insects‟ attack (Arimura et al., 2005; Wei et al., 2007). Volatiles produced by some green

leaves have been reported to reduce bioactivity and performance of herbivores and

sometimes have antifungal activity (Kaori et al., 2006).

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2.4 The Economic Importance of Floral Scent

Floral scent or flower fragrance is very important in perfumery, cosmetic, agricultural

and cut flower industries. Flower fragrance produced by flowering plants such as

jasmine, roses, and lavender are pleasant to human sensory system and have potential

application as perfume ingredients (Rees, 1991). The high demand on floral fragrances

for perfumery and food industries has caused researchers to focus on fragrance-related

biochemical compounds and their biosyntheses (Knudsen et al., 1993). The knowledge on

natural floral fragrances and their specific components are used in perfume production to

produce synthetic perfumes and mimic the natural floral fragrance (Verdonk et al., 2003).

An example of the highly commercialized floral fragrances in perfumery industry are

rose (Rosa hybrida) (Zuker et al., 1998; Guterman et al., 2002) and jasmine (Jasminum

grandiflorum) (Kaiser, 1993).

In agricultural industry, pollination of crops is very important for fruit development. The

highest yield can be obtained whenever the highest pollination occurs in field with the

help of pollinators. Domesticated crops from other parts of the world might not be

suitable for local pollinators due to drastic changes of morphology and biochemistry of

the plants (Pichersky and Dudareva, 2007). Commercialization of the plants might also

be prevented by the lack of natural pollinators. Domestication of natural pollinators of the

plant into other territory is also not usually successful due to the lack of ability of the

pollinators to adapt to the new environment (Buchmann and Nabhan, 1996). Scent

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engineering of local and new introduced plant species into new territory might enhance

pollination by local pollinators (Pichersky and Dudareva, 2007).

In cut-flower industry which is known as a multi-billion dollar industry, extensive work

on breeding of cultivated flowers to improve their vase life, shipping characteristics, and

visual aesthetic values such as shape and colour has contributed to the lost of their

original scent (Vainstein et al., 2001). Genetic engineering approach by transformation of

selected genes for the selected traits might restore the original scent in the plants. Besides

that, the production of scent in scentless flowering plants or modification of floral scent

can also be done by genetic engineering to increase the commercial values of the flower

in cut-flower industry (Pichersky and Dudareva, 2007).

2.5 Floral Scent and Its Volatile Compounds

The scent of scented flowers varies between species due to the combination of the

compositional and the level of each compound (Knudsen et al., 1993; Dudareva et al.,

2000). The entire floral organs are involved in floral scent emission but petal is the main

source of floral scent in most flowers (Pichersky et al., 1994). Floral scents are stored in

special oil glands such as trichome before released to the air as volatiles (Effmert et al.,

2006). Analysis on volatile compounds in floral scent by headspace with gas

chromatography-mass spectrometry (GC-MS) method has led to the discovery of more

than 1700 chemical structures (Knudsen and Gershenzon, 2006). Floral scent is a

complex mixture of low molecular mass molecules such as monoterpenes,

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sesquiterpenes, benzenoids, phenylpropanoids and fatty acid derivatives (Knudsen et al.,

1993). Besides that, there are also other compounds in floral scents such as nitrogen and

sulfur containing compounds including indole, a compound from amino acid metabolism

(Knudsen and Gershenzon, 2006).

In floral scent studies, more than 500 terpenoid compounds have been identified

including monoterpene (C10), sesquiterpene (C15), diterpenes (C20) and irregular terpenes

(Knudsen and Gershenzon, 2006). Examples of monoterpenoid compounds identified in

floral scent studies are linalool, ocimene, mycrene, nerol, citranellol and geraniol.

Linalool compound has been identified in floral scent of snapdragon (Antirrhinum majus)

(Nagegowda et. al, 2008), Clarkia breweri (Raguso and Pichersky, 1995), Arabidopsis

thaliana (Chen et al., 2003) and a lot of orchid species such as Dendobium beckleri,

Dendobium brymerianum, and Phalaenopsis violacea (Kaiser, 1993). Other

monoterpenoid compounds such as mycrene and ocimene were detected in the scent of

Anthirrinum majus (Dudareva et al., 2003), Nicotiana suaveolens (tobacco) (Raguso et

al., 2003), Arabidopsis thaliana (Chen et al., 2003) and also in some orchids such as

Platanthera chlorantha, Polystachya cultriformis and Zygopetalum crinitum (Kaiser,

1993).

Besides monoterpenoid compounds, sesquiterpenoids such as germacrene D, farnesene,

caryophyllene, copaene and nerolidol were also detected in floral scent of many plant

species. Germacrene D has been detected in the scent of Rosa hybrida (Hendel-

Rahmanim et al., 2007), Petunia hybrida (Verdonk et. al, 2003), and some orchids such

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as Aerangis confusa, Aerangis biloba, and Dendrochilum cobbianum (Kaiser, 1993).

Caryophyllene, another volatile sesquiterpene compound, was also detected in many

scented flowers including Arabidopsis thaliana (Chen et al., 2003), carnation (Dianthus

caryophyllus) (Schade et al., 2001), and some orchids such as, Cattleya lawrenceana,

Cattleya percivaliana, Dendrobium trigonopus and Dendrochilum cobbianum (Kaiser,

1993). Besides germacrene D and caryophyllene, nerolidol is another compound of

sesquiterpene found in scented flowers including Antirrhinum majus (Nagegowda et. al,

2008), Nicotiana suaveolens (tobacco) (Raguso et al., 2003) and also in some orchids

such as Diaphananthe pulchella, Epidendrum ciliare, Masdevallia estradea and

Zygopetalum crinitum (Kaiser, 1993).

The other class of volatile compounds that is highly distributed among scented flowers is

benzenoids and phenylpropanoids. So far, more than 300 volatile compounds of this class

have been identified in floral scent of plant species which include methylbenzoate,

methylsalicylate, phenylacetaldehyde, phenylethyl acetate, benzyl acetate, phenylethanol,

eugenol and isoeugenol (Knudsen and Gershenzon, 2006). Methylbenzoate has been

detected in some scented flowers such as Petunia hybrida (Verdonk et. al, 2003),

Antirrhinum majus (Nagegowda et. al, 2008), Stephanotis floribunda (Pott et al., 2002)

and also in some orchids such as Dendrobium trigonopus, Encyclia baculus and Laelia

perinii (Kaiser, 1993). Benzyl benzoate has been identified to be emitted by floral organs

of some scented flowers including Petunia hybrida (Verdonk et. al, 2003), Nicotiana

suaveolens (Raguso et al., 2003), Stephanotis floribunda (Pott et al., 2002) and also in

some orchids such as Dendrobium moniliforme, Dendobium monophyllum, Dendrobium

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williamsonii, and Dendrochilum cobbianum (Kaiser, 1993). Eugenol and isoeugenol are

compounds of benzenoid and phenylpropanoid classes which also contribute to the floral

scent of scented flowers including Petunia hybrida (Verdonk et. al, 2003), Clarkia

breweri (Raguso and Pichersky, 1995), Stephanotis floribunda (Pott et al., 2002) and also

some orchids such as Angraecum bosseri, Himantoglossum hircinum, Lycaste aromatica,

Phalaenopsis violancea, and Platanthera bifolia (Kaiser, 1993).

Other important class of floral scent compounds is fatty acid derivatives which are

derived from lipoxygenase pathway such as hexanol, hexanal, nonanal, pentadecane,

decanal and dodecanal (Knudsen and Gershenzon, 2006). Volatile of fatty acid

derivatives are normally detected in vegetative tissues and play an important role in plant

defense. Traces of fatty acid derived compounds have been detected in floral scent of

some scented flowers (Knudsen and Gershenzon, 2006). In the floral scent of Petunia

hybrida, some of the fatty acid derivatives that were detected included decanal,

dodecanal, 3-hexenal and 2-hexenal (Verdonk et al., 2003). Besides that, some orchids

were also reported to emit fatty acid derivatives such as octanal, 2-heptanol, nonanal,

decanal, methyl decanoate and ethyl decanoate (Kaiser, 1993; Hsiao et al., 2006).

2.6 The Fragrance Biosynthetic Pathway and Molecular Biology of Floral Scent

To date, many volatile compounds have been identified but only few enzymes and genes

involved in the fragrance biosynthetic pathways have been characterized. Therefore, the

mechanisms of fragrance formation are still not fully understood (Dudareva et al., 2000).

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Most of the volatile compound analyses have been done by using gas chromatography-

mass spectrometry (Guterman, 2002). For many years, research on floral scent was

focused on its chemical rather than biological aspects due to the complexity of the studies

involved in volatile emission (Vainstein et al., 2001). There are three major biosynthetic

pathways involved in floral scent production which are terpenoid, benzenoid/

phenylpropanoid and lipoxygenase pathways (Croteau and Karp, 1991). Common

modifications such as hydroxylation, acetylation and methylation have been described

even though the pathways leading to the final products have not been fully characterized

(Guterman et al., 2002).

Terpenoid compounds are synthesized via the terpenoid pathway (Figure 2) which is

localized in both the plastid and cytosol (McCaskill and Croteau, 1995; Lichtenthaler,

1999; Rohmer, 1999). There are two initial pathways in the terpenoid pathway;

Methylerythritol Phosphate (MEP) Pathway in the plastid (Lichtenthaler, 1999; Rohmer,

1999) and Mevalonic Acid (MVA) Pathway in the cytosol (McCaskill and Croteau,

1995). Both pathways play an important role in the production of dimethylallyl

diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP) (McCaskill and

Croteau, 1995; Lichtenthaler, 1999; Rohmer, 1999). Condensation of one DMAPP

molecule with one IPP molecule generates the production of geranyl diphosphate (GPP)

which is the main precursor for monoterpenes such as linalool, ocimene, and mycrene

(Ogura and Koyama, 1998; Poulter and Rilling, 1981) meanwhile condensation of a

DMAPP with two IPP molecules generates the production of farnesyl diphosphate (FPP)

which is the main precursor for sesquiterpenes such as caryophyllene, germacrene D and

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Figure 2: Terpenoid Biosynthetic Pathway in Plastid and Cytosol. This diagram was

adopted from Nagegowda et al., 2008.

Abbreviations: DMAPP, dimethylallyl diphosphate; GA-3P, glyceraldehydes-3-

phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; DXR, DXP reductoisomerase; MEP,

2-C-methyl-D-erythritol-4-phosphate; DXS, DXP synthase; FPP, farnesyl diphosphate;

FPPS, farnesyl diphosphate synthase; GPP, geranyl diphosphate; GPPS, geranyl

diphosphate synthase; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl

diphosphate synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, 3-

hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; MVA,

mevalonic acid.

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nerolidol (McGarvey and Croteau, 1995). At present, several fragrance-related genes,

cDNAs and enzymes responsible for the production of terpenoid have been isolated and

characterized from several plants such as linalool synthase from Clarkia breweri

(Pichersky et. al, 1995), Arabidopsis thaliana (Chen et. al, 2003), Antirrhinum majus

(Nagegowda et. al, 2008), ocimene synthase from Antirrhinum majus (Dudareva et. al,

2003), mycrene synthase from Antirrhinum majus (Dudareva et. al, 2003) and

germacrene D synthase from Rosa hybrida (Guterman et al., 2002).

Benzenoid and phenylpropanoid pathway (Figure 3) is another fragrance biosynthetic

pathway identified in the plant system besides the terpenoid pathway. Benzenoids and

phenylpropanoids are derived from phenylalanine as the main precursor (Gang et al.,

2001). The pathway starts with the deamination of L-phenylalanine to trans-cinnamic

acid catalyzed by L-phenylalanine ammonia lyase (PAL), followed by shortening of the

C2 unit of the side chain of cinnamic acid for formation of benzaldehyde compound via a

few proposed pathways such as CoA-dependent ß-oxidative, CoA-independent-non-ß-

oxidative pathway or a combination of these two pathways (Boatright et al., 2004). The

benzenoid pathway is proceeded with the oxidation of benzaldehyde to benzoic acid

which is the main precursor for the formation of volatile benzenoids and

phenylpropanoids such as benzylbenzoate, benzylacetate, methybenzoate, and

phenylethyl acetate (Boatright et al., 2004). There are other phenylpropanoids emitted in

scented plants such as eugenol, isoeugenol, and methyleugenol which are synthesized via

other routes without going through benzoic acid as an intermediate. Coniferyl alcohol and

coniferyl acetate play a role in this other routes as intermediates with cinnamic acid as the

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Figure 3: Benzenoid and Phenylpropanoid Biosynthetic Pathway. This chart was

adopted from Pichersky and Dudareva, 2007. It represents a compilation of reactions and

enzymes in several scented plants including Clarkia breweri and Petunia hybrida.

Abbreviations: BEAT, acetyl-coenzyme A:benzylalcohol acetyltransferase; BPBT,

benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase; BSMT, benzoic

acid/salicylic acid carboxylmethyltransferase; BZL, benzoate:CoA ligase; CFAT,

coniferyl alcohol acyltransferase; EGS, eugenol synthase; IEMT, S-adenosyl-L-

methionine:(iso)eugenol O-methyltransferase; IGS, isoeugenol synthase; PAAS,

phenylacetaldehyde synthase; PAL, phenylalanine ammonia-lyase; SAMT, salicylic acid

carboxyl methyltransferase.

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main precursor for the production of these compounds (Boatright et al., 2004: Pichersky

and Dudareva, 2007).

There are other volatile phenylpropanoid compounds derived from phenylalanine without

going through cinnamic acid such as phenylacetaldehyde and phenylethanol (Boatright

et. al, 2004). The pathway starts with the transamination of phenylalanine to

phenylpyruvate followed by decarboxylation to phenylacetaldehyde (Vuralhan et al.,

2003; Boatright et al., 2004). Reduction of phenylacetaldehyde produces phenylethanol

while oxidation of phenylacetaldehyde will lead to the production of an ester

phenylacetate (Erlich 1907; Vuralhan et al., 2003). Phenylacetaldehyde synthase (PAAS)

plays an important role in the decarboxylation of phenylalanine to phenylacetaldehyde

(Boatright et al., 2004; Kaminaga et al., 2006). PAAS has been reported to be a cytosolic

homotetradimeric enzyme that belongs to group II pyridoxal 5‟-phosphate-dependent

amino acid decarboxylase (Sandmeier et al., 1994). In floral scent studies, two PAAS

have been identified from Petunia hybrida (PhPAAS) and Rosa hybrida (RhPAAS) that

share 64% identity (Kaminaga et al., 2006). PhPAAS and RhPAAS were reported to

share ~50-60% identity with other plant decarboxylases such as tyrosine decarboxylases,

tryptophan decarboxylases and aromatic amino acid decarboxylases (Kaminaga et al.,

2006).

The third pathway related to fragrance biosynthesis is lipoxygenase pathway. The

products of this pathway are derived from C18 fatty acids (linoleic and linoleic acids)

which are cleaved into C6 and C12 components by hydroperoxide lyase (Feussner and

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Wasternack, 1998). Hydroperoxide lyase produces either 3-cis hexenal or hexanal which

are the common constituents of volatiles in green leaf or flower depending on the C18

substrate (Knudsen et al., 1993). This 3-cis hexenal or hexanal can be further converted

to alcohols (3-cis-hexenol or hexanol) or 3-hexenyl acetate (D‟Auria et al., 2002).

2.7 Floral Scent Studies on Orchids

Floral scent studies on orchids was initiated in early 1980s. However, early work was

focused on detection of volatile compounds emitted by orchid flowers using gas

chromatography-mass spectrometry (GC-MS) method (Kaiser, 1993). Identification of

floral scent compounds in orchids has led to extensive biochemical and molecular studies

of other scented plants such as Clarkia breweri and Petunia hybrida. However, the floral

scent biosynthetic pathways of orchids are still far from understood (Hsiao et al., 2006).

Identification of volatile compounds has been carried out on the scent of more than 180

orchid species and hybrids including Cattleya araguaiensis, Cymbidium formosanum,

Dendrobium carniferum, Dendrobium superbum, Oncidium curcutum, Phalaenopsis

violacea, Vanda tessellata (Kaiser, 1993) and Phalaenopsis bellina (Hsiao et al., 2006)

by GC-MS. Based on the GC-MS analysis on the orchids‟ scent, monoterpenoids and

sesquiterpenoids are the highly distributed compounds among orchid species. Some of

those include linalool, mycrene, ocimene, germacrene D and nerolidol. Benzenoids and

phenylpropanoid compounds that are also found in scented orchids include

methylbenzoate, benzyl benzoate, benzyl acetate, phenylethyl acetate, eugenol and

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isoeugenol. Besides that, there were traces of other compounds detected in scented

orchids such as fatty acid derivatives, indole and formanilide (Kaiser, 1993; Hsiao et al.,

2006).

In Vanda tessellata (one of the parents of Vanda Mimi Palmer), more than 20 volatile

compounds have been identified in its scent including linalool, mycrene, ocimene, methyl

benzoate, methyl isobutyrate, cinnamic aldehyde, cinnamic alcohol, methyl cinnamate,

benzyl acetate, phenylethyl acetate and indole. Methylbenzoate was the highest volatile

compound emitted by Vanda tesellata representing 61.5% of the total scent, followed by

linalool (23%), cinnamic aldehyde (5.1%) and methyl cinnamate (4.6%). Other minor

compounds identified in the scent of Vanda tessellata were methyl isobutyrate, methyl 2-

methylbutyrate, α-pinene, mycrene, ocimene, benzyl acetate, methyl salicylate, 3-

phenylpropanoid, cinnamic aldehyde, α-ionone, cinnamic alcohol and indole (Kaiser,

1993).

On the molecular biology aspect, only recently a group of researchers from Taiwan have

reported expressed sequence-tags (ESTs) on a scented orchid species Phalaenopsis

bellina (Hsiao et al., 2006). Isolation and identification of putative fragrance-related

cDNAs was achieved by comparing floral ESTs sequences of Phalaenopsis bellina with

ESTs of a non-scented orchid, Phalaenopsis equestris. From their work, the

monoterpenes biosynthetic pathway of linalool, mycrene and geraniol was elucidated

using the bioinformatics approach, Pathway and Literature (PAL) finder program. From

the ESTs of Phalaenopsis bellina, several fragrance-related cDNAs that encode for

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geranyl diphosphate synthase, epimerase, lipoxygenase, diacylglycerol kinase, O-

methytransferase, and cytochrome P450 monooxygenase were isolated. The only

fragrance-related cDNA which has been well characterized in orchids is geranyl

diphosphate synthase (PbGDPS) from Phalaenopsis bellina (Hsiao et al., 2008). Real-

time RT-PCR analysis has shown that the expression of PbGDPS gene increased

gradually once the bud open and reached the highest peak on the fifth-day after bud-

opening. After that, the expression decreased gradually until the end of the flower‟s life.

The same expression pattern was reported on the emission of monoterpene compounds

such as linalool and geraniol. Protein characterization has shown the PbGDPS enzyme is

a homodimeric enzyme which can catalyze the formation of both geranyl diphosphate

(GDP) and farnesyl diphosphate (FPP) which are the main precursors for the production

of monoterpenoids and sesquiterpenoids, respectively.

2.8 Floral Scent Studies in Vanda Mimi Palmer

Preliminary work on scent analysis of Vanda Mimi Palmer was carried out by nose

detection during day time from 8.00am to 5.00pm and at different floral developmental

stages (Janna et al., 2005). The study shows that emission was at the highest level

between 12-2pm when the flower was fully-open. Subsequent molecular biology work on

Vanda Mimi Palmer was focused on the construction of a floral cDNA library of Vanda

Mimi Palmer representing almost all mRNA transcripts of fully-open flower at different

time points in a 24-hour cycle and at different flower developmental stages such as early

bud (green), mature bud (red), half-open flower and fully-open flower (Chan, 2009; Chan

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et al., 2009).Two fragrance-related cDNAs were isolated from a preliminary sequencing

of 100 clones from the floral cDNA library. The transcripts are 1-deoxy-D-xylulose 5-

phosphate reductoisomerase (accession number: EU145744) (Chan et al., 2009) and

lipoxygenase (Chan, 2009). Besides that, a suppression subtraction hybridization (SHH)

was also carried out to isolate more fragrance-related cDNAs by hybridizing the

substracted open flower cDNA library with two different cDNA probes of open flower

and bud stage during day time separately. From the SSH work, another two fragrance-

related cDNAs were successfully isolated which are sesquiterpene synthase (accession

number: EU145743) and alcohol acyltransferase (accession number: EU145742) (Chan,

2009).

Molecular characterization has been carried out on the 1-deoxy-D-xylulose 5-phosphate

reductoisomerase, by subjecting it to a full-length cDNA isolation and gene expression

analysis by real-time RT-PCR. The expression studies were carried out in different

tissues, at different floral developmental stages and at different time points in a 24-hour

cycle. Besides that, other fragrance-related cDNAs such as sesquiterpene synthase and

alcohol acyltransferase were also characterized in the same manner (Chan, 2009).

Characterization of the fragrance-related transcripts showed upregulated expression in

floral tissues especially in the petal and sepal compared to vegetative tissues such as leaf,

shoot and root. Expression analyses of the fragrance-related transcripts at different

developmental stages and different time points in a 24-hour cycle has shown that

fragrance biosynthesis in Vanda Mimi Palmer is developmentally and rhythmically

regulated (Chan, 2009; Chan et al., 2009).

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CHAPTER 3

MATERIALS AND METHODS

3.1 Plant Material

Orchid plants (Vanda Mimi Palmer and Vanda Tan Chay Yan) used for this study were

purchased from the United Malaysian Orchids Sdn. Bhd., a nursery located in Rawang,

Selangor. The purchased plants were maintained in the nursery by Mr. Eric Kok. Both

Vanda Mimi Palmer and Vanda Tan Chay Yan used in this study were grown separately

in pots with charcoal under tropical climate (12 hours in light followed by 12 hours in

dark), temperature between 25-300C and exposed to 70-80% of sunlight. Samples for

volatile analysis such as flowers and buds were directly captured from the orchid plants

without detaching the flowers. For essential oil extraction, the flowers were directly

processed after being detached from the mother plants without freezing in liquid nitrogen.

Meanwhile, samples for RNA work such as flowers, buds, leaves, shoots and roots were

detached from the mother plant, frozen in liquid nitrogen and stored in -800C before use.

All of the plants used for volatile analyses, essential oil extraction as well as RNA

extraction were brought to Universiti Putra Malaysia and placed outside of laboratory

building with almost similar condition to the nursery at Rawang (not directly exposed to

the sun) to ensure growth as well as scent biosynthesis and emission are similar to when

the plants are grown in the nursery.

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3.2 Analysis of the Scent of Vanda Mimi Palmer by GC-MS

Determination of the constituents of the scent of Vanda Mimi Palmer was carried out by

gas chromatography-mass spectrometry (GC-MS) to identify the volatile compounds

emitted by Vanda Mimi Palmer at different developmental stages and also at different

time points in a 24-hour cycle. Emission analysis of the floral scent of Vanda Mimi

Palmer was carried out by GC-MS at three different floral developmental stages; bud,

half-open flower and fully-open flower. Temporal emission analysis was carried out by

GC-MS for every two hours interval (12am, 2am, 4am, 6am, 8am, 12pm, 2pm, 4pm,

6pm, 8pm and 10pm) in a 24-hour cycle to determine the emission pattern of each single

compound. Besides that, GC-MS analysis on open flower of Vanda Tan Chay Yan was

carried out in order to compare with the volatiles emitted by Vanda Mimi Palmer. All of

the volatile emission analyses were carried out in three replicates using flowers from

different mother plants. Average of the three replicates of the volatile analysis in a 24-

hour cycle was used to plot graphs with the error bar showing the standard error for the

three replicates.

Volatiles emitted by single flower were captured by Solid Phase Micro-Extraction

(SPME) (Supelco, USA). The SPME with silica fiber that was coated with 100µm

polydimethylsiloxane (PDMS) was used to absorb the volatiles emitted by the flowers of

Vanda Mimi Palmer and Vanda Tan Chay Yan. A single flower was put into a modified

funnel without detaching the flower from the flower stalk (Figure 4). The back of the

funnel was covered with aluminum foil. The SPME holder was pressed to allow the silica

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Figure 4: Solid Phase Micro Extraction (SPME) Used to Capture Volatile

Compounds Emitted by the Flower of Vanda Mimi Palmer. The flower was trapped

and captured by the SPME for 15 minutes.

Funnel

Silica fiber of SPME

SPME holder

Flower

Aluminium foil

Retort stand

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fiber in the SPME emerge from the SPME syringe and captured volatiles produced by the

flower for 15 minutes. After that, The SPME fiber was thermally desorbed for 1 minute

at 2500C in an injector port of Shimadzu GC-MS (Shimadzu, Japan) with the port in

splitless injector mode. Volatile compounds were separated by using a capillary HP-5

column (50m x 0.32mm, film thickness 1.05µm) with helium (21 kPa) as a carrier gas.

The GC oven was programmed 450C for 1 minute followed by an increase of 10

0C per

minute to 2800C. The temperature 280

0C was extended for 10 minutes. Mass spectra of

the eluted compounds were recorded for the m/z value of 30-300. The spectrum given by

each compound was compared to the National Institute of Standards and Technology

(NIST) spectral library 2002 (Scientific Instrument Services, USA).

3.3 Extraction and Analysis of Essential Oil of Vanda Mimi Palmer

Essential oil was extracted from Vanda Mimi Palmer by soaking 100 grams of open

flower of Vanda Mimi Palmer in 800ml hexane for 48 hours. After soaking, the debris

was removed from hexane with essential oil by filtering with 1MM Whatman filter paper

(GE healthcare, USA). The essential oil was recovered by evaporating in a 250 ml round

bottom flask with a neck size of 24/29 using a rotary evaporator machine with water bath

set at 450C. This step was repeated until all of the hexane was evaporated from the

essential oil. The weight of the round bottom flask was recorded before and after the

evaporation process by the evaporator machine. After that, the essential oil in paste form

was dissolved in 5ml acetone and then adjusted to 10 part per million (ppm). A volume of

1.5µl of the 10ppm essential oil was analyzed by GC-MS using the same protocol as

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described in section 3.2. The same approach was applied to the flowers of Vanda Tan

Chay Yan.

3.4 Isolation of Total RNA

The RNA extraction procedure was based on Yu and Goh method (2000) with minor

modifications (Chan et al., 2009). One gram samples of Vanda Mimi Palmer from

different tissues/organs (petal, sepal, leaf, root and shoot), at different floral

developmental stages (young bud (green), mature bud (red), half-open flower, fully-open

flower, and 14-day old flower) and at different time points in a 24 hour cycle (12am,

2am, 4am, 6am, 8am, 12pm, 2pm, 4pm, 6pm, 8pm and 10pm) were ground saperately

into fine powder in liquid nitrogen. The ground samples were mixed thoroughly with

20ml of pre-warmed (650C) extraction buffer [100mM Tris-Cl (pH 7.5) containing 20mM

EDTA (pH 8), 2M NaCl, 2% (w/v) hexadecyl (cetyl) trimetyl ammonium bromide

(CTAB), 1% (w/v) polyvinylpyrrilidone (PVP), and 2% (v/v) β-mercaptoethanol]. The

mixture was then incubated at 650C for 15 minutes followed by centrifugation at 12,857 x

g, 40C for 15 minutes to pellet the cellular debris. After the centrifugation, recovered

supernatant was mixed thoroughly with an equal volume of chloroform:iso-amylalcohol

(24:1). The mixture was shaken vigorously followed by centrifugation at 12,857 x g, 40C

for 15 minutes. After the centrifugation, chloroform:iso-amylalcohol (24:1) extraction

was carried out twice to recover the top aqueous phase. Lithium chloride (LiCl) was then

added to the recovered top aqueous phase to a final concentration of 2M followed by

incubation at 40C overnight. Recovered RNA was then pelleted by centrifugation at

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12,857 x g, 40C for 30 minutes. The RNA pellet was rinsed in cold 80% (v/v) ethanol and

air-dried before dissolving in 100µl diethyl pyrocarbonate (DEPC)-treated water. The

RNA samples were then stored at -200C until further use but no longer than two months.

The quantity and quality of the total RNA were determined by spectrophotometric

readings at 230nm, 260nm, and 280nm using the Biophotometer (Eppendorf, Germany)

and nanospectrophotometer (Implen, Germany). The integrity of RNA was checked by

formaldehyde denaturing agarose gel electrophoresis (see section 3.4.1).

3.4.1 Formaldehyde Denaturing Agarose Gel Electrophoresis

Formaldehyde denaturing agarose gel of 1.2% (w/v) was prepared by melting 0.4 gram

agarose powder in 30ml of 1X F buffer [20mM MOPS buffer (pH 7) containing 1mM

EDTA and 5mM NaOAc]. The melted agarose was allowed to cool to 500C and

formaldehyde was added to a final concentration of 6% (v/v). The formaldehyde agarose

gel was then poured into a gel cast and allowed to solidify. While waiting for the gel to

be solidified, 1µg of total RNA was added into sample buffer [1X F buffer containing

50% (v/v) formamide and 6% formaldehyde (v/v)] followed by addition of a final

concentration of 0.24X loading dye [0.25%(w/v) bromophenol blue, 0.25% xylene

cyanol, 30% (v/v) glycerol] and 1µg of ethidium bromide. The sample was then

incubated at 650C for 10 minutes and placed on ice immediately before loading into the

gel. Gel electrophoresis was carried out in a formaldehyde running buffer [1X F buffer

containing 6% (v/v) formaldehyde] at 45 Volts for 1 hour. The agarose gel was destained

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for 30 minutes in autoclaved DEPC-treated water and viewed using a gel documentation

system (Bio-Rad, USA).

3.5 Isolation of PolyA+

mRNA

The PolyATract® mRNA Isolation Systems III (Promega, USA) was used to isolate the

polyA+ mRNA from total RNA. One miligram of total RNA was made up to a final

volume of 500µl by addition of nuclease-free water provided in the kit. The RNA sample

was then incubated at 650C for 10 minutes. Annealing reaction was then carried out by

adding 150pmol biotinylated oligo(dT) probe and Sodium Saline Citrate (SSC) buffer,

pH 7 to a final concentration of 0.5X (diluted from 20X SSC provided in the kit) to the

RNA solution for the oligomers to anneal with polyA+ mRNA. The mixture was then

mixed gently and allowed to completely cool at room temperature.

While waiting for the RNA sample to completely cool, streptavidin-paramagnetic

particles (SA-PMPs) (provided in the kit) were washed by gently flicking the bottom of

the SA-PMPs‟ tube until the particles were completely dispersed. The SA-PMPs were

then captured by placing the tube in a specific magnetic stand (provided with the kit) for

about 30 seconds until the SA-PMPs were completely accumulated on the wall of the

tube. Recovered supernatant was carefully removed and the SA-PMPs particles were

washed three times with 300µl of 0.5X SSC buffer (diluted from 20X SSC provided in

the kit), each time being captured by magnetic stand as described above. The washed SA-

PMPs particles were finally resuspended in 100µl of 0.5X SSC buffer.

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The total RNA from the annealing reaction was then added to the washed SA-PMPs. The

mixture was then incubated at room temperature for 10 minutes and mixed gently for

every 2 minutes. The SA-PMPs particles were then captured again as described above

and the recovered supernatant was carefully removed without disturbing the SA-PMPs

particles. The SA-PMPs particles were washed four times with 300µl of 0.1X SSC buffer

(diluted from 20X SSC provided in the kit). Each washing was carried out by gently

flicking the bottom of the tube until the SA-PMPs particles were completely dispersed.

The final supernatant was removed as much as possible without disturbing the SA-PMPs

particles.

These steps were followed by elution of polyA+

mRNA from the recovered SA-PMPs

particles. The SA-PMPs particles were resuspended by gently flicking in 100µl of RNAse

free water. The SA-PMPs particles were then magnetically captured and the eluted

mRNA was transferred into a sterile RNAse free microcentrifuge tube. The elution step

was repeated by resuspending the SA-PMPs pellet in 200µl of RNAse free water. The

recovered polyA+

mRNA was precipitated by addition of a final concentration of 0.3M

sodium acetate (NaOac) pH 5.2 and 80% (v/v) isopropanol. The precipitation was carried

out at -200C overnight. After the overnight incubation, the mixture was centrifuged at

12,857g at 40C for 20 minutes. Recovered mRNA pellet was washed with 75% (v/v)

ethanol and air-dried. The mRNA pellet was then dissolved in 20µl RNAse-free water

and stored in -200C for subsequent use. The quantity and purity of the eluted polyA

+

mRNA was determined by spectrophotometric readings using a Biophotometer

(Eppendorf, Germany).

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3.6 Double-stranded cDNA Synthesis

Universal Riboclone®

cDNA synthesis system (Promega, USA) was used for double-

stranded cDNA synthesis from mRNA isolated in section 3.5. There are two major parts

involved in the cDNA synthesis which are first-strand cDNA synthesis and second-strand

cDNA synthesis. First-strand cDNA synthesis was carried out by addition of 1µg of oligo

(dT) into 2µg of mRNA sample. Nuclease-free water (provided in the kit) was added up

to a total volume of 15µl. The mixture was then incubated at 700C for 10 minutes and

placed on ice immediately followed by addition of 40 units of RNAsin® and 5µl of 5X

first-strand buffer (provided in the kit) into the mixture. The mixture was heated at 420C

for 5 minutes followed by addition of 30 units of AMV reverse transcriptase and sodium

pyrophosphate to a final concentration of 2mM. The mixture was then incubated at 420C

for 60 minutes. The first-strand cDNA synthesized was then used as template for the

synthesis of second-strand cDNA.

Second-strand cDNA synthesis was carried out by adding 40µl of 2.5X second-strand

buffer (provided in the kit), 5µg of acetylated Bovine Serum Albumin (BSA), 25 units of

DNA polymerase I, and 0.8 units of RNAse H into the synthesized first strand-cDNA.

Nuclease-free water was then added up to a total volume of 100µl followed by incubation

at 140C for two hours. The double-stranded cDNA synthesized was then heated at 70

0C

for 10 minutes to stop the cDNA synthesis. The double-stranded cDNA was then placed

on ice and 0.2 unit of T4 DNA polymerase was added into the mixture followed by

incubation at 370C for 10 minutes. After the incubation, EDTA with a final concentration

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of 20mM was added to stop the reaction. Phenol-chloroform extraction was then carried

out by addition of equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1) into the

synthesized double-stranded cDNA. The mixture was then vortexed briefly, followed by

a centrifugation at 15,871 x g at room temperature for 1 minute. After the centrifugation,

recovered aqueous phase was precipitated by adding sodium acetate to a final

concentration of 0.3M (pH5.2) and two volumes of cold absolute ethanol (pre-chilled at

40C). The precipitation was carried out at -80

0C for 30 minutes. Centrifugation at 15,871

x g for 15 minutes was then carried out at 40C to pellet the pure double-stranded cDNA.

After centrifugation, the supernatant was discarded and the recovered double-stranded

cDNA in pellet form was rinsed with 70% (v/v) ethanol. The pellet was then air dried,

dissolved in 20µl of Tris-EDTA (TE) buffer pH 8 and then stored at -200C until further

use.

3.6.1 Quantification of Double-stranded cDNA

The quantity of the double-stranded cDNA synthesized was determined by ethidium

bromide plate assay. A volume of 30ml of 0.8% (w/v) agarose gel was prepared in 1X

Tris-acetate-EDTA (TAE) buffer, pH 8. The agarose was melted and allowed to cool to

~500C. Ethidium bromide to a final concentration of 0.1µg/ml was added into the molten

agarose. The agarose mixture was mixed well by swirling and then poured into a 100 mm

Petri dish. A few columns and lanes were plotted at the back of the Petri dish before the

agarose solution containing ethidium bromide was poured into it. The agarose solution

was allowed to solidify.

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39

Standard DNA (Lambda DNA) (Fermentas, Canada) used in the ethidium bromide assay

were diluted in nuclease-free water. The concentrations of standard used for the ethidium

bromide plate assay were 200ng/µl, 150ng/µl, 100ng/µl, 75ng/µl, 50ng/µl, 25ng/µl,

10ng/µl and 5ng/µl. The standards and the synthesized double-stranded cDNA in section

3.6 were spotted onto the solidified ethidium bromide plate. Both standards and cDNA

spotted on the plate were allowed to be absorbed by the agarose placed in a fume hood

for about 20 minutes. The ethidium bromide assay was viewed using a gel documentation

system (Bio-Rad, USA). The concentration of the cDNA sample was determined by

comparing the signal intensity produced by the cDNA with the standard.

3.7 cDNA Library Screening

A floral cDNA library of Vanda Mimi Palmer previously constructed by Miss Chan Wai

Sun (Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia) was used for this study. The cDNA library was constructed by

using the Zap-cDNA® Gigapack

® III Gold Cloning Kit (Stratagene, USA). The cDNA

library represents all mRNAs expressed in Vanda Mimi Palmer‟s flower at all different

flowering stages in a 24-hour cycle. The titer of the cDNA library provided was 7.8 x 109

pfu/ml. In this study, the floral cDNA library was hybridized with fully-open flower

cDNA probe of Vanda Mimi Palmer containing pool of mRNA transcripts expressed by

fully-open flower during daylight hours (8am, 10am, 12pm, 2pm, 4pm and 6pm). The

clones that gave positive signals were selected as putative fragrance-related cDNA

candidates for further characterization.

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40

3.7.1 Probe Labeling

The double-stranded cDNAs synthesized in section 3.6 was used as templates for probe

preparation. The double-stranded cDNA was labelled with biotin by using the NEBlot®

Phototape® Kit (New England BioLabs Inc, USA) according to the manufacturer‟s

instruction. The protocol of probe labeling used was based on random priming labeling

method. The probe labeling was carried out with 1µg double-stranded cDNA as template

and nuclease-free water was added to a total volume of 34µl. A control reaction was

prepared by using 1µg of unbiotinylated lambda DNA (Hind III digested lambda DNA)

(provided in the kit) as the template. The same condition was applied for the control

reaction. Incubation at 970C for 5 minutes was then carried out to break the hydrogen

bond between the double-stranded cDNA. The denatured double-stranded cDNA was

then placed on ice for 5 minutes followed by the addition of 10µl of 5X labelling buffer

containing biotinylated random octamers (provided in the kit), 5 units of klenow

fragment (3‟-5‟exo-) and dNTPs mix a with final concentration of 5mM of dTTP, 5mM

dGTP, 5mM dCTP, and 5mM of the mixture of dATP and biotynilated-dATP (Biotin-14-

dATP) (provided in the kit) and the mixture was incubated at 370C for 20 hours. The

reaction was then terminated by the addition of EDTA (pH 8) to a final concentration of

20mM. Purification was then carried out by adding two volumes of cold absolute ethanol

and 10M lithium chloride (LiCl) to a final concentration of 0.14M and a precipitation

step was then carried by incubation at -800C for 30 minutes. The mixture was centrifuged

at 15,871 x g, at 40C for 15 minutes to pellet the synthesized probe. The pellet was then

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41

washed with 70% (v/v) ethanol. After air dried, the pelleted probe was dissolved in 20µl

of TE buffer and stored at -200C for further use.

3.7.1.1 Detection of Labeling Efficiency

The probe synthesized was subjected to a serial dilution of 1/5, 1/52, 1/5

3, 1/5

4, and 1/5

5

in nuclease-free water in order to check the labeling efficiency of the probe. One

microliter of each diluted probe was spotted onto a Hybond-N+ nylon membrane

(Amersham Bioscience, UK) and air dried for 10 minutes. The membrane was then

soaked in 0.4N NaOH for 2 minutes followed by three times agitation in 2X SSC buffer

for 2 minutes. The membrane was then air dried before continuing with SDS detection

method. The SDS detection method was initiated by agitating the membrane in a washing

buffer [2.5mM sodium phosphate buffer, pH 7.2 containing 0.5% (w/v) sodium dodecyl

sulphate (SDS) and 12.5mM NaCl] for 5 minutes. The membrane was then agitated in

10ml blocking solution [25mM sodium phosphate buffer, pH 7.2 containing 5% (w/v)

SDS and 125mM NaCl] for 15 minutes at room temperature. The blocking solution was

then added with streptavidin-alkaline phosphatase conjugate (Promega, USA) at a

dilution factor of 1/10,000 and agitated for 10 minutes. The blocking solution was then

discarded and the membrane was washed twice with washing buffer. The first wash was

carried out for 5 minutes while the second wash for 1 hour. The washing buffer was then

discarded and the membrane was equilibrated in 10ml of detection buffer [20mM Tris-Cl

pH 9.5 containing 20mM NaCl and 2mM MgCl2] for 5 minutes. Then, the membrane was

transferred into a transparent plastic bag and was spread evenly with 200µl of ready to

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42

use CDP-star® (Roche, USA), a chemiluminescent substrate. The CDP-star

® was allowed

to equilibrate for 2 minutes and the excess CDP-star and bubbles were removed by

rolling a glass rod on the plastic bag. The plastic bag was then sealed, placed in an X-ray

cassette followed by an exposure to an X-ray film (Kodak, USA) for 30 minutes. After

the exposure, the film was developed in a developer solution (Kodak, USA). The X-ray

film was rinsed in distilled water, followed by fixation in a fixer solution (Kodak, USA)

in a dark room to visualize the signals on the X-ray film.

3.7.2 Primary Screening

In this study, 500,000 pfu of the floral cDNA library was plated onto 10 NZY (Appendix

A) agar plates (150 mm petri dishes) with XL1-Blue MRF cells, a recombinant

Escherichia coli host strain provided in the Zap-cDNA® Gigapack

® III Gold Cloning Kit

(Stratagene, USA). Plaques were allowed to grow to hairpin size for 8 hours at 370C. The

plaques were then transferred onto a 147 mm diameter plaque lift nylon membrane

(Amersham Biosciences, UK) and hybridized with the fully-open flower cDNA probe.

The clones that give positive signals on X-ray film were cored out and used for secondary

screening.

3.7.2.1 Preparation of Bacterial Culture for Infection.

A loop of the XL1-Blue MRF cells from a glycerol stock was streaked onto LB agar

(Appendix A) containing 12.5µg/ml tetracycline. The plate was incubated overnight at

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43

370C. After overnight incubation, a colony of the XL1-Blue MRF was picked and

inoculated into 5ml of LB broth (Appendix A) with supplements [10mM MgSO4, 0.2%

(w/v) maltose]. The culture was incubated at 370C overnight in an incubator shaker,

shaking at 200rpm. The overnight bacterial culture was then subcultured into 50ml LB

broth with supplements and incubated at 370C in the incubator shaker, shaking at 200rpm

until the OD600 reached ~0.5. The bacterial culture was then transferred into two tubes

equally and centrifuged at 12,857 x g at room temperature for 2 minutes to pellet the

bacterial cells. After the centrifugation, the bacterial pellet was resuspended in half of the

original volume with 10mM MgSO4 and the OD600 was then adjusted to 0.5 for infection

purpose.

3.7.2.2 Preparation of Filter for Primary Screening

A serial dilution of the floral cDNA library of Vanda Mimi Palmer was carried out in SM

buffer [50mM Tris-Cl, pH 7.5 containing 100mM NaCl, 8mM MgSO4.7H2O and 0.002%

(w/v) gelatin] from the original stock to produce a titer of 780pfu/µl for plating purpose.

The diluted cDNA library in a volume of 64.1µl (~50,000 pfu) was mixed with 600 µl of

XL1-Blue MRF cells (OD600 = 0.5) and incubated at 370C for 15 minutes to allow phage

particles in the cDNA library to attach to the XL1-Blue MRF cells. NZY top agarose (see

Appendix A) at ~370C in a volume of 6.5ml was mixed immediately with the mixture in

a 15ml sterile centrifuge tube and then poured onto a NZY plate. The plate was swirled

quickly to spread the NZY top agar evenly on the NZY plate before it solidified. The

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44

plate was then incubated at 370C in an incubator for 8 hours to allow the formation of

plaques. The plate was then stored at 40C at least for 2 hours prior to plaque lift.

3.7.2.3 Transferring the Plaques onto Membrane (Plaque Lift)

Commercial plaque lift Hybond-N+, nylon membrane (Amersham Biosciences, UK) with

a diameter of 147 mm was used to transfer the plaques formed on NZY agar plates. The

membranes were marked by cutting at three sites asymmetrically. A cut membrane was

initially placed onto the plate containing plaques for 2 minutes and the cut sites were

marked at the bottom of the plate. A second plaque lift was repeated for the same plate by

placing another membrane for 4 minutes, as a duplicate. The membranes were then air

dried for 15 minutes. Each of the membrane (plaque side up) was placed onto a 3MM

Whatman paper (GE healthcare, USA) pre-wetted with a denaturing solution [1.5M

NaCl, 0.5M NaOH] for 2 minutes followed by a neutralization solution [0.5M Tris-Cl

buffer, pH 8 containing 1.5M NaCl] for 5 minutes. The membranes were then rinsed in a

rinsing solution [0.2M Tris-Cl buffer, pH 7.5 containing 2X SSC) for 25 seconds in the

same condition. The membranes were then air-dried for 30 minutes and then baked at

800C for 2 hours. The NZY agar plates were kept at 4

0C up to two weeks for further use.

3.7.2.4 Pre-hybridization and Hybridization of Membrane

Hybridization was carried out based on the instruction manual of Phototape® -Star

Detection Kit (New England Biolabs Inc., USA) with modifications. Firstly, baked nylon

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45

membranes were pre-wetted in a 2X SSC buffer for 2 minutes. The membranes were then

transferred into 15ml of pre-warmed (650C) pre-hybridization buffer and pre-hybridized

in a HB-1000 Hybridization machine (Techne, UK) for 30 minutes at 550C. While pre-

hybridizing, a mixture of 1µg of biotin-labeled open-flower cDNA probe, 80µg of

sheared salmon sperm DNA and 10X SSC buffer was denatured in boiling water for 10

minutes. After denaturing, the probe mixture was added into the pre-hybridization

solution in a hybridization bottle and hybridization was carried out at 550C for 16 hours.

After 16 hours of hybridization, the membranes were washed twice in low stringency

condition with 2X washing solution [2X SSC, 0.1% SDS] by agitation at room

temperature for 5 minutes. The 2X washing solution was discarded, followed by high

stringency wash with 0.5X washing solution [0.5X SSC, 0.1% (w/v) SDS] at 550C for 15

minutes. The high stringency wash was repeated for 30 minutes with fresh 0.5X washing

solution in the same manner. After the high stringency wash, the membranes were

equilibrated in washing buffer [2.5mM sodium phosphate buffer, pH 7.2 containing 0.5%

(w/v) SDS and 12.5mM NaCl] for 5 minutes. Blocking step was then carried out by

agitating the membranes in blocking solution [25mM sodium phosphate, pH 7.2

containing 5% (w/v) SDS and 125mM NaCl] for 15 minutes at room temperature. The

membranes were then agitated in fresh blocking solution with streptavidin-alkaline

phosphatase conjugate (Promega, USA) at a dilution factor of 1/10,000 for 15 minutes at

room temperature. The membranes were then washed twice with washing buffer [2.5mM

sodium phosphate, pH 7.2 containing 0.5% (w/v) SDS and 12.5mM NaCl]. The first

wash was carried out for 5 minutes while the second wash for 60 minutes. The

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46

membranes were then equilibrated in detection buffer [20mM Tris-Cl pH 9.5 containing

20mM NaCl and 2mM MgCl2] for 10 minutes. The membranes were then transferred into

a transparent plastic bag and 500µl of ready to use CDP-star® (Roche, USA), a

chemiluminescent substrate was added and spread evenly on the membrane. The CDP-

star® was allowed to equilibrate for 2 minutes and the excess CDP-star and bubbles were

removed by rolling a glass rode on the plastic bag. The plastic bag was then sealed,

placed in an X-ray cassette followed by an exposure to an X-ray film (Kodak, USA)

overnight. After the exposure, the film was developed as described in section 3.7.1.1.

3.7.2.5 Coring of Positive Clones

A light box was used to align back the plates with the positive signals on X-ray film.

Each of the positive plaques was cored out into a fresh 1.5ml microcentrifuge tube

containing 500µl SM buffer and 20µl chloroform. The tubes were then vortexed briefly

and incubated at room temperature for 30 minutes. After that, the tubes were stored at

40C for further use.

3.7.2.6 Secondary Screening

A set of serial dilution (10-1

, 10-2

, 10-3

, 10-4

, and 10-5

) was prepared for the cored out

phages from primary screening in SM buffer. A volume of 20µl of each phage dilution

was mixed with 350µl of XL1-Blue MRF cells (OD600=0.5) in a fresh 1.5ml sterile

microcentrifuge tube separately. The mixtures were then incubated at 370C for 15

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47

minutes to allow the phage particles to attach onto the bacterial cells. Subsequently, the

mixtures were resuspended with 3.5ml of ~370C NZY agarose top agar and immediately

poured onto NZY agar plates. The plates were swirled evenly before the agarose

solidified, sealed with parafilm and incubated at 370C for 16 hours. The plates were then

kept at 40C for at least 2 hours before transferring the plaques onto membrane. The

dilution of 10-3

was chosen to be applied for all of the clones cored out in primary

screening because this dilution gave 50 to 100 plaques on each NZY agar plate (100 mm

petri dish).

Plaque lifting was carried out in the same manner as described in the primary screening

in section 3.6.2.3 with 87mm diameter immobilon membranes (Millipore, USA). The

subsequent steps such as pre-hybridization, hybridization, washing and detection were

carried out in the same condition as mentioned in section 3.7.2.4. Positive plaque from

each plate that represents each positive clone was cored out and resuspended with 500µl

SM buffer and 20µl chloroform in a sterile 1.5ml microcentrifuge tube. The tube was

then vortexed briefly and incubated at room temperature for at least 30 minutes before

storing at 40C for further use.

3.8 Single Clone In Vivo Excision

Phagemids of the putative positive clones were subjected to in vivo excision. Initially,

SOLR cells and XL1-Blue MRF cells were cultured in 5ml LB broth (see Appendix A)

with supplement [10mM MgSO4, 0.2% (w/v) maltose] separately. The culture was

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48

incubated at 300C with shaking at 200 rpm for 16 hours. The 5ml overnight culture was

sub-cultured into 50ml of LB with supplement. The culture was incubated at 300C with

shaking at 200rpm until the OD600 reached 0.5. The tube containing the culture was then

centrifuged at 1000g for 10 minutes to pellet the bacterial cells. The pellet was then

resuspended in 25ml of 10mM MgSO4. The OD600 of the resuspended bacterial cells was

then adjusted to 1.0.

In vivo excision was initiated by addition of 200µl of XL1-Blue MRF cells (OD600 =1.0),

250µl of phage (stock from secondary screening in SM buffer with chloform) and 1µl of

helper phage into 1.5ml microcentrifuge tube followed by incubation at 370C for 15

minutes to allow attachment of phage to XL1-Blue MRF cells. The mixture was then

added into 3ml of LB broth with supplements in a 15ml centrifuge tube followed by

incubation at 370C with shaking at 200rpm for 16 hours. After the incubation period, the

cultures were heated at 700C for 20 minutes followed by a centrifugation at 1000 x g for

15 minutes. After the centrifugation, recovered filamentous phages in supernatants were

transferred into fresh microcentrifuge tubes and stored at 4 0C for further use.

Plating of the excised phagemid was carried out by mixing 100µl of filamentous phages

of each clone with 400µl of SOLR cells (OD600 = 1.0) separately. The mixture was then

incubated at 370C for 15 minutes followed by plating of 200µl of the mixture on LB plate

containing 100µg/ml of ampicillin separately. The plate was incubated at 370C for 16

hours. After the incubation period, a single colony grown on the plate was re-streaked on

a fresh LB plate containing 100µg/ml of ampicillin and incubated at 370C for 16 hours.

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49

The single colony was used for colony PCR reaction, plasmid mini preparation and also

for glycerol stock preparation. The same procedure was applied for all of the clones cored

out in secondary screening.

3.9 Reverse-Northern Analysis

Reverse-Northern analysis was carried out in this study to select putative positive clones

that show up-regulated expression in fully-open flower stage compared to bud stage of

Vanda Mimi Palmer as candidates for fragrance-related cDNAs. Reverse-Northern

applied Southern Blotting method whereby PCR product of cDNA inserts were used in

the experiment instead of genomic DNA.

Firstly, colony PCR amplification of cDNA insert from each clone was performed. Single

colony of each clone grown on LB-ampicillin (100µg/ml) agar plates were picked out

using a tooth pick and swirled into PCR mix [1X PCR buffer (Invitrogen, USA), 1.5mM

MgCl2 (Invitrogen, USA), 0.2µM T3 universal primer (5‟-ATTAACCCTCACTAAAG-

3‟), 0.2µM T7 universal primer (5‟-AATACGACTCACTATAG-3‟), 0.2mM dNTPs

(Bioron, Germany), 1 unit Taq DNA polymerase (Invitrogen, USA)]. The PCR

conditions were as follow: 1 cycle of pre-denaturation at 950C for 5 minutes, followed by

35 cycles of denaturation at 950C for 15 seconds, annealing at 55

0C for 45 seconds and

extension at 720C for 1 minute, followed by final extension at 72

0C for 5 minutes. The

PCR amplification was carried out by using MJ Cycler and Master Cyler (Bio-Rad,

USA). The PCR products were then loaded in duplicate on 1.5% (w/v) agarose gel and

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50

electrophoresis was carried out at 80 Volts for 40 minutes. The gels were stained in

1µg/ml ethidium bromide solution for 1 minute followed by de-staining in distilled water

for 30 minutes. The gels were then viewed using a gel documentation system (Biorad,

USA). After viewing using the gel documentation system, the gels were used for

Southern Blotting (Sambrook and Russell, 2001).

In Southern Blotting, the gels were denatured in 0.4 N NaOH (denaturing solution) for 15

minutes and blotted onto a Hybond-N+ nylon membranes (Amersham Bioscience, UK)

through gravitational transfer overnight in 0.4 N NaOH (Sambrook and Russell, 2001).

After the overnight transfer, the wells position on agarose gels were marked on the

membranes by using a pencil. The membranes were then air dried and baked at 800C for

2 hours. The baked membranes were then hybridized with two different probes

separately: open flower cDNA probe and bud cDNA probe. The open flower and bud

cDNA probes were synthesized separately using NEBlot® Phototape Kit (New England

BioLabs Inc, USA) as described in section 3.7.1. The hybridization, washing and

detection steps were same as mentioned in section 3.7.2.4. The clones that showed

stronger signals with open flower probe compared to bud probe of Vanda Mimi Palmer

were selected for partial sequencing and further characterization.

3.10 Elimination of Cymbidium mosaic Virus Coat Protein cDNA Transcripts

In preliminary sequencing of 100 clones of the floral cDNA library by Miss Chan Wai

Sun, half of the clones in the floral cDNA library were reported to be contaminated by

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51

Cymbidium mosaic virus coat protein cDNA transcript. The clones that represent the

Cymbidium mosaic virus coat protein cDNA transcript (see Appendix B) were eliminated

from the screened clones (section 3.9) by hybridizing the PCR-amplified inserts of all the

in vivo excised clones with Cymbidium mosaic coat protein cDNA probe. The

membranes used in reverse-Northern analysis in section 3.9 were stripped off by using

hot-SDS method as described in the manual of Hybond+

nylon membrane (Amersham

Bioscience, UK). Boiled 0.1% SDS (w/v) was poured onto each membrane in a tray and

was agitated for 10 minutes. The stripped membranes were re-hybridized with

Cymbidium mosaic virus coat protein cDNA probe as described in section 3.7.1.

The template used for probe synthesis was PCR-amplified from plasmid containing

Cymbidium mosaic virus coat protein cDNA transcript. The amplification was carried out

using gene specific primers (CMV-F 5‟-AGAGCCCACTCCAACTCCA-3‟ and CMV-

R 5‟-GCAGGCAGAGCATAG AGACTG-3‟) that amplified a partial cDNA sequence

(650bp) of the Cymbidium mosaic virus coat protein. The PCR reaction was carried out in

a final volume of 20µl containing PCR mix [1X PCR buffer (Invitrogen, USA)

containing 1.5mM MgCl2 (Invitrogen, USA), 0.2µM CMV-F primer, 0.2µM CMV-R

primer, 0.2mM dNTPs (Bioron, Germany) and 1 unit Taq DNA polymerase (Invitrogen,

USA)] and 100ng plasmid as initial template. The PCR conditions were as follow: 1

cycle of pre-denaturation at 940C for 3 minutes, followed by 35 cycles of denaturation at

940C for 30 seconds, annealing at 53

0C for 45 seconds and extension at 72

0C for 45

seconds, and a final extension at 720C for 5 minutes.

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52

3.11 Plasmid Mini Preparation

The clones that showed up-regulated expression in fully-open flower stage compared to

bud stage of Vanda Mimi Palmer were subjected to plasmid mini preparation as

described in Sambrook et al., 1989 with minor modifications. Initially, single colonies of

in vivo excised clones were inoculated in 3ml LB broth containing 100µg/ml ampicillin.

The cultures were then incubated in an incubator shaker at 370C, shaking at 200rpm for

16 hours. After the incubation period, the cultures were centrifuged at 15,294 x g for 1

minute at room temperature. The supernatants were then discarded and the recovered

pellets were resuspended in 200µl of Solution I [25mM Tris-Cl buffer pH 8 containing

10mM EDTA] followed by incubation on ice for 10 minutes. After that, 400µl of

Solution II [0.2N NaOH, 0.5% (w/v) SDS] was added into each tube and mixed gently by

inverting the tubes for several times followed by 10 minutes incubation on ice. After that,

300µl of Solution III [3M sodium acetate pH 4.8] was added into the tubes and mixed

completely. The tubes were then incubated on ice for 15 minutes followed by

centrifugation at 15,294 x g for 15 minutes at 40C. The supernatant was transferred into

fresh microcentrifuge tube and 50µg RNAse A was added into the tube. Removal of

RNA from plasmid DNA by RNAse A was carried out by incubation at 370C for 30

minutes.

After the incubation with RNAse A, 400µl of phenol: chloroform: isoamylalcohol

(25:24:1) was added into the tube. The mixture was then vortexed completely and

centrifuged at 15,294 x g at room temperature for 10 minutes. After that, top aqueous

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53

phase was transferred into new fresh microcentrifuge tube followed by precipitation.

Plasmid DNA was precipitated by adding 0.1 volume of 3M NaOAc, pH 5.2 and 0.6

volume of isopropanol and this was followed by incubation at -200C for 1 hour. The

plasmid DNA was pelleted by centrifugation at 15,294 x g at 40C for 20 minutes. The

supernatant was discarded and the pellet was rinsed with 70% (v/v) ethanol. The pellet

was air dried and dissolved in 20µl Tris-EDTA (TE) buffer, pH 8.

3.12 DNA Sequencing and Sequence Analysis

All of the clones that showed up-regulated expression in open flower stage compared to

bud stage of Vanda Mimi Palmer after the removal of Cymbidium mosaic virus coat

protein transcripts were sequenced using T3 primer. The sequencing reaction was carried

out by Medigene Sdn. Bhd. and First Base Sdn. Bhd., Kuala Lumpur, Malaysia. Vector

and adaptor sequences were eliminated before proceeding with sequence analysis. All the

sequences were subjected to contigs and singletons analyses using the CAP3 software

(http://pbil.univ-lyon1.fr/cap3.php) (Huang and Madan, 1999). The tentative unique

genes were compared against the GeneBank non-redundant database at the National

Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) using

BLASTX and BLASTN with a cut off value less than 1e-05

and score more than 80. The

Expressed Sequence-tags (ESTs) were then classified into functional groups based on

their putative functionality (Zhao et al., 2006; Lindqvist et al., 2006).

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54

3.13 Verification of Fragrance-related cDNA by RT-PCR Analysis

Total RNA samples for the verification of fragrance-related cDNA was extracted from

flowers and buds collected at 12.00 noon separately using the same protocol as described

in section 3.4. The total RNA samples were used as template for first strand cDNA

synthesis. PolyA tail mRNAs in the total RNA were reverse-transcribed into first strand

cDNA by using Quantitect reverse transcription kit (Qiagen, Germany) according to the

manufacturer‟s instruction. Initially, 1µg of total RNA template and 2µl of 7X of gDNA

wipe buffer (provided in the kit) were transferred into a clean PCR tube followed by

addition of RNAse-free water to a total volume of 14µl. The mixture was then incubated

at 420C for 2 minutes and chilled on ice quickly. The remaining components of the kit

were then added into the mixture which include reverse transcriptase enzyme (1µl),

Quantiscript RT buffer (4µl), and RT primer mix (1µl). The mixture was then incubated

at 420C for 30 minutes followed by incubation at 95

0C for 3 minutes to inactivate the

reverse transcriptase enzyme. The first strand cDNA for each sample was aliquoted into a

few microcentrifuge tubes and kept at -200C for storage.

Forward and reverse primers (Table 1) of each selected transcript were designed using

Primer 3 software with advance setting including the size of primers between 18-22bp,

melting temperature (Tm) of the primers were between 50-600C and the product size

ranged between 150-250bp. The primers were synthesized by First Base Sdn. Bhd.,

Malaysia. Gradient RT-PCR was carried out for each primer set for each clone of interest

containing cDNA insert to get the best annealing temperature. The gradient RT-PCR

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55

Table 1: Characteristics of Primers Used in Verification. Primers for putative

fragrance-related transcripts and endogenous control that were used in RT-PCR.

Target/ Amplicon

Length (bp)

Primers

Primer Sequences

Optimal

Annealling

Temperature

VMPCyP450

(201bp)

Forward

Reverse

5‟ GCTGTTTTCATGTCTGGAAGC 3‟

5‟ TCCTGTTTGTGACGGCTCTT 3‟

530C

VMPEST (182 bp)

Forward

Reverse

5‟ GCAACGCTCTCATGGTTTAT 3‟

5‟ AAAAGCCTCGAAAAATCTGA 3‟

570C

VMPCMEK

(202bp)

Forward

Reverse

5‟ GTACACGGAAACGATCACTG 3‟

5‟ AACATGCAAGCCAAACATT 3‟

590C

VMP36

(227bp)

Forward

Reverse

5‟- CTCCCGCATTGACCATAAAT-3‟

5‟-GGAACCACACCCAAACTCTC-3‟

550C

VMP48

(200bp)

Forward

Reverse

5‟-TTGGATGTCGTGAAGGCAAT-3‟

5‟- CAACACAAGAAGATAGCACAGCA-3‟

530C

VMP59

(221bp)

Forward

Reverse

5‟-CGAGGAAGACGAAGAGGAAG-3‟

5‟-CGAAAAATAGAACAGAGCCATAG-3‟

530C

VMP83

(202bp)

Forward

Reverse

5‟-GCTGGTTAGGGTGAAGCAT-3‟

5‟-AAAAACATAGACAAATGGAGACC-3‟

530C

VMP90

(243bp)

Forward

Reverse

5‟-GGAAAGGAAGAAAAGCAGCA-3‟

5‟-CGACACCAAGAAACATCTCC-3‟

590C

VMP96

(231bp)

Forward

Reverse

5‟-TCGCCTTCTCTCATCTCTGAA-3‟

5‟- CAAGCCCACGCATAAAAGTA-3‟

590C

VMPA28

(202bp)

Forward

Reverse

5‟-GTACACGGAAACGATCACTG-3‟

5‟-AACATGCAAGCCAAACATT-3‟

590C

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56

Target/ Amplicon

Length (bp)

Primers

Primer Sequences

Optimal

Annealling

Temperature

VMPA46

(182bp)

Forward

Reverse

5‟-GGATGTTCTACGGGTGGAC-3‟

5‟-AGAGAGGAGCACAGCTTTATTT-3‟

590C

VMPA54

(180bp)

Forward

Reverse

5‟-AAAAGCAGCGGTTTATGAAG-3‟

5‟-CCAAACGAAAACTCAGGAAT-3‟

530C

Elongation factor

(507bp)

-endogenous control

Forward

Reverse

5‟ CCGACCGCAAGGAGAGTTAT 3‟

5‟ AAGCCACGGAACAAAAACAG 3‟

550C

Page 57: Vanda Mimi Palmer Thesis

57

conditions were as follow: 1 cycle of pre-denaturation at 940C for 3 minutes, followed by

35 cycles of denaturation at 940C for 30 seconds, annealing at 50-60

0C for 45 seconds

and extension at 720C for 45 seconds, and a final extension at 72

0C for 5 minutes. The

best annealing temperature for each primer set for each sequence of interest was chosen

based on the highest amount (most intense band) of PCR-amplified products viewed

using a gel documentation system (Biorad, USA) with minimum or without primer

dimers.

After determining the optimum annealing temperature, RT-PCR was carried out using the

same PCR protocol as described above with specific annealing temperature (see Table 1

for the annealing temperature used) for each sequence of interest. The RT-PCR products

were analyzed by agarose gel electrophoresis separately and viewed using a gel

documentation system (Biorad, USA). After viewing, the agarose gels with RT-PCR

product for each clone were transferred onto nylon membranes separately by Southern

blot method as described in section 3.9. The membranes were baked at 800C for 2 hours.

The membranes were then hybridized with specific probe labeled with biotin representing

each cDNA of gene of interest. The membranes were hybridized for 16 hours with

specific probe separately followed by washing and detection steps as described in section

3.7.2.4. Putative elongation factor, a housekeeping gene cDNA transcript was obtained

from Miss Chan Wai Sun and used as an endogenous control for the RT-PCR reaction.

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The probes used for the hybridization were synthesized using NEBlot®

phototape kit as

described in section 3.7.1. The cDNA inserts of each clone were PCR-amplified using T3

(5‟-ATTAACCCTCACTAAAG-3‟) and T7 (5‟-AATACGACTCACTATAG-3‟)

universal primers prior to the probe synthesis. The PCR-amplified cDNA inserts of each

clone were purified using SpinPrep PCR clean-up kit (Novagen, Germany) according to

the manufacturer‟s instruction. A volume of 100µl of PCR product was mixed completely

with 400µl of SpinPrep bind buffer A (provided in the kit). The mixture was transferred

into a SpinPrep PCR filter with a receiver tube and centrifuged at 15,294 x g for 1

minute. The flow through was discarded and 400µl of the SpinPrep bind buffer A was

transferred into the filter and centrifuged at 15,294 x g for 1 minute. The PCR-amplified

insert at the surface of the filter‟s membrane was washed with 500µl of SpinPrep wash

buffer B (provided in the kit). The wash step was carried out by a centrifugation at 15,294

x g for 1 minute. The flow through was discarded and the filter was centrifuged again for

2 minutes to remove excess of wash buffer. After that, 30µl of pre-warmed (700C)

SpinPrep elution buffer C (provided in the kit) was transferred into the filter and allowed

to stand for 3 minutes at room temperature. The filter was transferred into a fresh 1.5ml

microcentrifuge tube and centrifuged at 15,294 x g for 1 minute. The same procedure was

applied to all the clones selected for the verification of putative fragrance-related

transcripts.

3.14 Full-length cDNA Isolation of Fragrance-related Transcripts

Full-length cDNA isolation was carried out for three selected putative fragrance-related

cDNAs from the verified transcripts in section 3.13 [putative 4-(cytidine 5′-diphospho)-2-

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59

C-methyl-d-erythritol kinase (VMPCMEK), putative cytochrome p450 protein transcript

(VMPCyP450) and an unknown protein transcript (VMPA28)] and a putative fragrance-

related cDNA transcript (putative phenylacetaldehyde synthase (VMPPAAS) isolated by

Miss Chan Wai Sun from the floral cDNA library of Vanda Mimi Palmer.

The cDNA templates for full-length cDNA isolation were synthesized using the SMART

RACE cDNA Amplification Kit (Clonetech, USA) according to the manufacturer‟s

instruction. The templates used for full-length cDNAs isolation were the 5‟- and 3‟-

RACE-Ready cDNAs synthesized from total RNA of Vanda Mimi Palmer at open flower

stage collected during day time at 12.00 noon. PCR amplification of 5‟- region was

carried out by using 5‟- RACE-Ready cDNA while 3‟- RACE-Ready cDNA was used as

template for PCR amplification of 3‟- region. For 5‟-RACE-Ready cDNA, 1µg of total

RNA sample was mixed with 1.7 μM of 5‟-CDS primer A (5'–(T)25V N–3' ) (N = A, C,

G, or T; V = A, G, or C) and 1.7 μM of SMART II A oligo (5'–

AAGCAGTGGTATCAACGCAGAGTACGCGGG–3'). Meanwhile for 3‟-RACE-Ready

cDNAs preparation, 1µg of total RNA sample was mixed with 1.7 μM of 3‟-CDS primer

A (5'–AAGCAGTGGTATCAACGCAGAGTAC(T)30V N–3' ) (N = A, C, G, or T; V =

A, G, or C). The tubes for 5‟-RACE-cDNA and 3‟-RACE cDNA were incubated at 700C

for 2 minutes followed by chilling on ice before addition of 1X first-strand buffer

(provided in the kit), 2mM of DTT (provided in the kit), 1mM of dNTP mix (provided in

the kit) and 1μl of MMLV reverse transcriptase (provided in the kit) for each tube. The

tubes were incubated at 420C for 1.5 hours to synthesize first-stand cDNA. After the

incubation period, 100µl of tricine-EDTA buffer (provided in the kit) was added into

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60

each of the 5‟-RACE and 3‟-RACE first-strand reaction products. The 5‟- and 3‟-RACE-

Ready cDNAs were then heated at 720C for 7 minutes to inactivate the reverse-

transcriptase enzyme. The 5‟- and 3‟-RACE-Ready cDNAs were then stored at -200C.

PCR amplification of 5‟- and 3‟-regions of the selected putative fragrance-related cDNA

transcripts was carried out by using the Advantage 2 Polymerase Mix (Clonetech, USA)

with gene specific primer for each transcript (Table 2) and universal primer mix (UPM)

provided in the SMART RACE cDNA amplification kit (Clonetech, USA) according to

the manufacturer‟s instruction with minor modifications. The 5‟ cDNA fragments of

VMPCMEK, VMPPAAS, VMPCyP450 and VMPA28 were PCR-amplified in 50µl PCR

reaction separately with 1X Advantage 2 PCR buffer (provided in the kit), 2.5μl of 5‟-

RACE-Ready cDNA, 1X Universal primer A mix (UPM) (see Table 2), 0.2 μM of gene

specific primer (listed in Table 2), 0.2mM of dNTP mix, and 1X Advantage 2

Polymerase Mix. The gene specific primers were designed manually at the conserved

region of the partial sequence of the cDNA transcript by considering the high melting

temperature (Tm) in the range of 65-720C with high GC content especially at the 3‟ end

of the primer, the size of the primers designed ranged between 25-30bp and synthesized

by Bioneer, Korea. The PCR amplification of 5‟- cDNA fragments were carried out by

pre- denaturation at 940C for 1 minute followed by 35 cycles of denaturaton at 94

0C for

30 seconds, annealing at 680C for 30 seconds and extension at 72

0C for 3 minutes. Final

extension was carried out at 720C for 5 minutes. The 3‟ cDNA fragment of VMPCyP450

and VMPA28 cDNAs were amplified by using the 3‟-RACE-Ready cDNA as the

template in the same manner as the 5‟- RACE PCR.

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Table 2: Primers Sequences for the Isolation of Full-length Transcripts. Primers of

fragrance-related transcripts were designed and used to isolate the 5‟-, 3‟-ends and ORF.

All of the primers except for UPM primers were synthesized by Bioneer, Korea.

Meanwhile UPM primers were provided in the Advantage2 polymerase mix (Clonetech,

USA).

Target/

Amplicon

Length (bp)

Primers

Primer Sequences

Optimal

Annealling

Temperature

VMPCMEK

5‟ region

VMPCMEK 5‟ GSP

UPM (long primer)

UPM (short primer)

5‟-GACCCTGTGGACGAAGTTGGCTCTG-3‟

5‟-CTAATACGACTCACTATAGGGCAAGCAGT

GGTATCAACGCAGAGT-3‟

5‟-CTAATACGACTCACTATAGGGC-3‟

680C

VMPCMEK

ORF

VMPCMEK ORF

forward

VMPCMEK ORF

reverse

5‟- CGC TTC TCA GCT TTT CTC CTA ACA ATG

GCC T -3‟

5‟- TTT TCC TGT TTG TGA CGG CTC TTC TCT

GCT CA -3‟

680C

VMPPAAS

5‟ region

VMPPAAS 5‟ GSP

UPM (long primer)

UPM (short primer)

5‟- AGCGTGAATCTTCTTGGTACAACCACCTC

-3‟

5‟-CTAATACGACTCACTATAGGGCAAGCAGT

GGTATCAACGCAGAGT-3‟

5‟-CTAATACGACTCACTATAGGGC-3‟

680C

VMPPAAS

ORF

VMPPAAS ORF

forward

VMPPAAS ORF

reverse

5‟- GAACTCCAGAAAATGGGCAGCCTTCCCA

C-3‟

5‟- CTCAGAGTGTTTTGAGTTTCCAACCCAGC

T-3‟

680C

VMPCyP450

5‟ region

VMPCYP450 5‟ GSP

UPM (long primer)

UPM (short primer)

5‟-CAGTAGTGTCCCTCCCTGCAATAACA-3‟

5‟- CTAATACGACTCACTATAGGGCAAGCAGT

GGTATCAACGCAGAGT-3‟

5‟-CTAATACGACTCACTATAGGGC-3‟

680C

VMPCyP450

3‟ region

VMPCYP450 5‟ GSP

UPM (long primer)

UPM (short primer)

5‟-TGAGAGCTAAACAAAACGGGCATCA 3‟

5‟- CTAATACGACTCACTATAGGGCAAGCAGT

GGTATCAACGCAGAGT-3‟

5‟-CTAATACGACTCACTATAGGGC-3‟

680C

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Target/

Amplicon

Length (bp)

Primers

Primer Sequences

Optimal

Annealling

Temperature

VMPCyP450

ORF

VMPCyP450 ORF

forward

VMPCyP450 ORF

reverse

5‟-GCTGCCACTAATGTCTTCTTCCTCAAGCTC

C-3‟

5‟- GTCTGCACTTCACTTATGGACAACAAACA

GAC-3‟

680C

VMPA28

5‟ region

VMPA28 5‟ GSP

UPM (long primer)

UPM (short primer)

5‟-AAATATCCCGCAACCTGTCCCACCT-3‟

5‟-CTAATACGACTCACTATAGGGCAAGCAGT

GGTATCAACGCAGAGT-3‟

5‟-CTAATACGACTCACTATAGGGC-3‟

680C

VMPA28

3‟ region

VMPA28 3‟ GSP

UPM (long primer)

UPM (short primer)

5‟-TGCCGAGCATTTGATGGACGAAAGT-3‟

5‟-CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3‟

5‟-CTAATACGACTCACTATAGGGC-3‟

680C

VMPA28

ORF

VMPA28 ORF

forward

VMPA28 ORF

reverse

5‟-TGTGAGATTAGTTCAATTCTTAGGCACC

CCAG-3‟

5‟-GCTTGTAGACAGCAACATGCAAGCCAAA

CATT-3‟

680C

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3.14.1 Purification of RACE-PCR Products

The RACE-PCR products were purified by using GeneAll Exspin Combo GP (GeneAll

Biotechnology, Korea) according to the manufacturer‟s instruction. The RACE-PCR

products were electrophoresed on 1.0% (w/v) agarose gel and in 1X TAE buffer, pH 8

(see Appendix A) at 80 Volts for 30 minutes. The gel was viewed using a gel

documentation system (Bio-Rad, USA). The band that showed expected size of PCR

product was excised using ethanol-cleaned razor blade. The excised gel slice was

weighed and transferred into a new fresh tube and 300µl of GB buffer (provided in the

kit) was added for each 100mg of the agarose gel slice. The mixture was incubated at

500C for 10 minutes and vortexed for every 2-3 minutes. The mixture in a volume of

700µl was then transferred into SV column (provided in the kit) and centrifuged at

15,294 x g for 1 minute. The flow through was discarded and the step was repeated until

finished all the mixture. After that, 500µl of fresh GB buffer was applied to the SV

column and centrifuged at 15,294 x g for 30 seconds. A volume of 700µl of washing

buffer (Buffer NW) (provided in the kit) was applied into the SV column and centrifuged

at 15,294 x g for 30 seconds. The flow through was discarded and the SV column was

centrifuged at 15,294 x g for 2 minutes to remove the excess washing buffer. The SV

column was then transferred into a fresh new 1.5ml microcentrifuge tube and 30µl of

elution buffer (Buffer EB) (Provided in the kit) was transferred into the SV column and

allowed to stand for 1 minute. A final centrifugation was carried out at 15,294 x g for 1

minute to recover the purified RACE-PCR product. The same procedure was applied for

purification of all RACE-PCR products in this study.

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3.14.2 Preparation of Competent Cells

Competent cells were prepared by calcium chloride treatment using Escherichia coli

DH5α strain (Sambrook et al., 1989). A single colony of DH5α from an LB (see

Appendix A) agar plate was inoculated into 5ml of LB broth (see Appendix A). The LB

broth culture was incubated at 370C overnight in an incubator shaker with shaking at

200rpm. A volume of 2ml of the overnight culture was transferred into 20ml fresh LB

broth in a 50ml centrifuge tube and incubated in the incubator shaker at 370C with

shaking at 200rpm for 2 hours. After the incubation period, the tube was centrifuged at

1000 x g for 5 minutes at 40C. The pellet was resuspended in 10ml of 75mM cold

calcium chloride (CaCl2) followed by incubation on ice for 20 minutes. After the

incubation period, the bacterial cells were centrifuged at 1000 x g for 5 minutes at 40C.

The pellet was resuspended in 2ml of 75mM cold CaCl2, followed by addition of 15%

(v/v) glycerol. A volume of 100µl of competent cells with 15% (v/v) glycerol was

aliquoted into 1.5ml microcentrifuge tubes. The stocks were kept at -800C for future use.

3.14.3 Cloning and Transformation of RACE-PCR Product

The purified RACE-PCR product in section 3.14.1 was cloned into the yTA cloning

vector (Yeastern, Taiwan) according to the manufacturer‟s instruction. An amount of

150ng of the purified RACE-PCR product was transferred into a 0.2ml PCR tube

followed by addition of ligation components provided in the kit; 1µl of ligation buffer A,

1µl of ligation buffer B, 50ng of yT&A cloning vector, and 1unit of yT4 DNA ligase.

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Nuclease-free water was then added up to a total volume of 10µl. The ligation reaction

was carried out at 40C overnight.

The ligation product was transformed into Escherichia coli DH5α competent cells

prepared in section 3.14.2 by heat shock transformation method (Sambrook et al., 1989).

A volume of 2.5µl of the ligation product was transferred into the 100µl of competent

cells followed by incubation on ice for 20 minutes. After the incubation period, a heat-

shock treatment was carried out by incubating the tube at 420C for 90 seconds followed

by incubation on ice for 1 minute. A volume of 1ml of LB broth (see Appendix A) was

added into the tube. The tube was then incubated at 370C with shaking at 250rpm for 45

minutes. After that, the tube was centifugated at 15,294 x g for 1 minute and 800µl of

supernatant was discarded. The pellet was then resuspended in the remaining supernatant.

A volume of 100µl of the resuspended DH5α cells was spreaded onto LB plate

containing 2.5mg of ampicillin (see Appendix A) with 0.8mg of X-gal and 1.4mg of

IPTG for blue and white screening. The plate was then incubated at 370C incubator oven

for 16 hours.

After the 16 hours incubation period, the transformants (white colonies) were re-streaked

onto a fresh LB plate containing 2.5mg of ampicillin. The plate was then incubated at

370C incubator oven for 16 hours. A single colony of each transformant was subjected for

colony PCR reaction with M13 forward (5' GTAAAACGACGGCCAGT 3') and M13

reverse (5' GCGGATAACAATTTCACACAGG 3') universal primers to select for a

transformant with the exact insert size. The colony PCR was carried out as described in

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66

section 3.8 except for the universal primers used were M13 forward and reverse instead

of T3 and T7. The clone with the expected size of PCR product was subjected for

plasmid mini preparation (see section 3.11) and sent for sequencing with both M13

forward and reverse primers. The same procedure was applied for all 5‟- and 3‟- RACE-

PCR products.

3.14.4 Isolation of Open Reading Frame (ORF) of the Putative Fragrance-related

Transcripts

The open reading frame (ORF) of VMPCMEK, VMPPAAS, VMPCyP450 and VMPA28

were isolated by PCR-amplification using the gene specific primers (see Table 2). The

amplifications were carried out in 50µl reaction containing 0.2µM forward primer (see

Table 2), 0.2µM reverse primer (see Table 2), 1mM of dNTP mix (Clonetech, USA),

2.5μl of 5‟-RACE-Ready cDNA and 1X Advantage 2 Polymerase Mix (Clonetech,

USA). The gene specific primers were designed manually at the conserved region of the

partial sequence of the cDNA transcript by considering the high melting temperature

(Tm) in the range of 65-720C with high GC content especially at the 3‟end of the primers.

The size of the primers ranged between 30-34bp and synthesized by Bioneer, Korea. The

PCR cycling parameters used were as follow; pre-denaturation at 940C for 1 minute

followed by 35 cycles of denaturation at 940C for 30 seconds, annealing at 68

0C for 30

seconds and extension at 720C for 3 minutes. Final extension was carried out at 72

0C for

5 minutes. The PCR products were electrophoresed on 1.0% (w/v) agarose gel in 1X

TAE buffer (see Appendix A) at 80 Volts for 30 minutes. The gel was viewed using a gel

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documentation system (Bio-Rad, USA). The band that showed the expected size of PCR

product was excised using ethanol-cleaned razor blade. The PCR products were purified

by using GeneAll Exspin Combo GP (GeneAll Biotechnology, Korea) according to the

manufacturer‟s instruction (see section 3.14.1). The purified PCR products were cloned

into yT&A cloning vector (see section 3.14.3) and transformed into Escherichia coli

DH5α competent cells (see section 3.14.3). A transformed colony was grown overnight

for plasmid mini preparation (see section 3.11) and sent for sequencing. The sequencing

was carried out by Macrogen Inc, Korea.

3.14.5 Sequence Analysis of the Cloned ORF

The ORF sequences of the putative fragrance-related cDNAs were analyzed by using

BLASTX program (NCBI GeneBank) (http://www.ncbi.nlm.nih.gov) to search for the

homologous sequences. Bioedit software version 7.0.1 (Hall, 1999) was used to translate

the cDNA sequences of the putative fragrance-related transcripts into amino acid

sequences. The amino acid sequences were then analyzed by BLASTP program (NCBI

GeneBank) (http://www.ncbi.nlm.nih.gov). Clustal W multiple alignment program

applying BLOSUM62 matrix in the Bioedit software was used to align the amino acid

sequences of the putative fragrance-related transcripts with homologous sequences from

other plants based on default setting. Expasy program was used to compute the

theoretical pI and predict molecular weight (MW) of the proteins (Expasy)

(http://br.expasy.org/tools/). Phylogenetic trees were constructed using the Mega version

4 software (Tamura et al., 2007) with Neighbour-Joining method to determine the genetic

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68

relationship between the deduced proteins of putative fragrance-related transcripts with

closely related proteins available in NCBI GenBank database. Unrooted trees were

produced with 1000 sample replication. Motifs search and signal peptide prediction was

carried out by Localizome software (http://localodom.kobic. re.kr/LocaloDom/index.htm)

(Lee et al., 2006), and Expasy tool (http://br.expasy. org/tools/).

3.15 Expression Studies of Fragrance-related Transcript by Real-time RT-PCR

Expression analysis of the putative fragrance-related transcripts (VMPPAAS,

VMPCMEK, VMPCyP450 and VMPA28) was carried out by real-time RT-PCR in

different tissues, at different developmental stages and different time points in a 24-hour

cycle. For expression studies in different tissues, total RNA was isolated from both floral

(bud, open-flower, petal, sepal and lip) and vegetative tissues (leaf, shoot, root and stalk).

For expression studies at different flower developmental stages, total RNA was isolated

from young bud (green), mature bud (red), half-open flower, fully-open flower and 14-

day old fully-open flower. All the samples for expression analysis on different tissues and

developmental stages were collected at 12.00pm in the afternoon since the scent emission

of Vanda Mimi Palmer was detected at high level at this time. For expression studies at

different time points in a 24-hour cycle, fully open flowers were collected for every 2

hours starting at 8.00 am until 6.00 am on the next day. The procedure used for total

RNA extraction from all the above mentioned samples was as described in section 3.4.

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For expression analysis, first-strand cDNA was synthesized from 1µg of total RNA using

Quantitect reverse transcription kit (Qiagen, Germany) as described in section 3.13. Real-

time RT-PCR reaction was carried out by using Brilliant® SYBR

® Green QPCR master

mix (Stratagene, USA) with 1µl of 10X diluted cDNA template with gene specific

primers (150nM forward primer and 150nM reverse primer). The primers used for the

real-time expression analysis were designed at the 3‟ end. The primers were designed

using Primer 3 software with advanced setting including size of primers in the range of

20-25bp, melting temperature (Tm) between 55-650C and the product size between 150-

250bp. The primers were synthesized by Sigma-Aldrich, USA. The primers‟ sequence

used for expression studies of gene of interest VMPPAAS, VMPCMEK, VMPCyP450,

and VMPA28 are listed in Table 3. Four replicates were prepared for each sample and

they are known as technical replicates. Besides that, 4 replicates of negative control

known as non-template control (NTC) were included for each real-time RT-PCR reaction

using the same mastermix with the gene of interest without any template.

3.15.1 Optimization for Real-time RT-PCR

Gradient real-time RT-PCR was carried out to select the best annealing temperature for

each putative fragrance-related transcript without any primer dimers. Gradient real-time

RT-PCR was carried out by using iQ5 cycler (Biorad, USA) utilizing the following

program: 950C for 10 min; 40 cycle of 95

0C for 30 sec, 55-65

0C for 30 sec (10

0C

temperature range) and 720C for 30 sec; 81 cycles for melting curve analysis; 10 sec for

each 0.50C (55-95

0C). Melt curve analyses were carried out for genes of interest and

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Table 3: Primers Sequences for the Real-time RT PCR. Primers were designed at 3‟

end of the gene of interest and were synthesized by Sigma-Aldrich, USA. The primers

were used to analyze the expression of each putative fragrance-related transcript in

different tissues, at different developmental stages and different time points in a 24-hour

cycle.

Target/ Amplicon

Length (bp)

Primers

Primer Sequences

Annealing

Temperature

VMPCMEK

(201bp)

Forward

Reverse

5‟-GCTGTTTTCATGTCTGGAAGC-3‟

5‟-TCCTGTTTGTGACGGCTCTT-3‟

590C

VMPPAAS Forward

Reverse

5‟-ACGAAATGTTGGGAAATGAATA-3‟

5‟-ACTTGCCTTCTCTTGAACCA-3‟

590C

VMPCyP450

(182 bp)

Forward

Reverse

5‟-GCAACGCTCTCATGGTTTAT-3‟

5‟-AAAAGCCTCGAAAAATCTGA-3‟

570C

VMPA28

(202bp)

Forward

Reverse

5‟-GTACACGGAAACGATCACTG-3‟

5‟-AACATGCAAGCCAAACATT-3‟

590C

Endogenous

control /

Amplicon length

(bp)

Actin

(236bp)

Forward

Reverse

5‟-CAGTGTTTGGATTGGAGGTTC-3‟

5‟- CCAGCAGCAGTCAGGAAAA-3

590C

Alpha tubulin (227bp)

Forward

Reverse

5‟- CTCCCGCATTGACCATAAAT-3‟

5‟-GGAACCACACCCAAACTCTC-3‟

560C

Cyclophilin

(200bp)

Forward

Reverse

5‟-TTGGATGTCGTGAAGGCAAT-3‟

5‟-

CAACACAAGAAGATAGCACAGCA-3‟

590C

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71

endogenous controls to check for sequence specificity amplification (see Appendix H).

The gradient real-time RT-PCR reaction was also carried out for each reference genes

(endogenous controls) using the same protocol as described above for the putative

fragrance-related transcript. The endogenous controls selected for the expression studies

were actin (Accession no: AF246716), alpha tubulin (Accession no: GW687608)

and cyclophilin (Accession no: GU942927). The optimal annealing temperature for

transcripts of interest and housekeeping genes are listed in Table 3.

PCR Amplification Efficiency Test

PCR amplification efficiency test was performed on all putative fragrance-related

transcripts and reference genes as decribed in the Guide to Performing Relative

Quantification of Gene Expression Using Real-time Quantive PCR (Applied Biosystems,

2004). First strand cDNA of fully-open flower of Vanda Mimi Palmer was used for PCR

amplification efficiency test. The cDNA was diluted in a series of five fold (50, 5

-1, 5

-2,

5-3

, and 5-4

). The PCR amplification was carried out utilizing the following program:

950C for 10 min; 40 cycle of 95

0C for 30 sec, annealing for 30 sec (see Table 3) and

extension at 720C for 30 sec; 81 cycles for melting curve analysis; 10 sec for each 0.5

0C

(55-950C). A standard curve was plotted against the log input of template cDNA amount

and the CT value of each dilution. The PCR efficiency was estimated by the slope of

standard curve (Equation A, in Appendix C), calculated using Equation B (Appendix C).

The accepted PCR efficiency is ranged between 90-110% and slope in the range of 3.1-

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3.6 as decribed in the Guide to Performing Relative Quantification of Gene Expression

Using Real-time Quantive PCR (Applied Biosystems, 2004).

3.15.3 Expression Analysis of Putative Fragrance-related Transcripts

The relative quantity (Q) of cDNA transcript of each gene was calculated by the software

using Equation C (see Appendix C). Normalization expression was carried out by

normalizing the relative cDNA quantity of each gene of interest with cDNA of reference

genes (endogenous control) using Equation D (see Appendix C) where the relative

quantity of all reference genes was used for normalization factor (see Equation E in

Appendix C). Finally, the normalized expression level of the target gene was rescaled

with the calibrator using Equation F (see Appendix C).

For expression studies at different tissues and developmental stages, bud was used as

calibrator while for relative expression studies at different time points, the relative

quantity of the transcripts at 12.00am was used as calibrator. The calibrator for each

expression analysis was selected as a reference tissue for comparative amount of

transcript in each tissue studied whereby the expression of the calibrator is equal to 1.

Up-regulated expression refers to the relative amount of the transcript in the tissue that is

more than 1 compared to the calibrator while down-regulated expression refers to the

relative amount of the transcript in the studied tissue that less than 1 in comparison to the

calibrator.

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Expression of each putative fragrance-related transcript was analyzed using iQ5 software

(Biorad, USA) by applying ∆∆CT comparative method. The ∆∆CT Comparative method

was used to estimate the relative expression level of the fragrance-related transcripts. The

normalized expression results were plotted in graphs for the relative quantity of the

transcripts in different tissues, at different developmental stages and different time points

in a 24-hour cycle compared to the calibrator.

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CHAPTER 4

RESULTS

4.1 Biochemical Analysis on Flowers of Vanda Mimi Palmer

4.1.1 GC-MS Analysis of the Scent of Vanda Mimi Palmer

Floral scent compounds of Vanda Mimi Palmer were captured by solid phase micro-

extraction (SPME) and were determined by Gas chromatography-mass spectrometry

(GC-MS). GC-MS analysis (Figure 5 and Table 4) shows that the scent of Vanda Mimi

Palmer was dominated by terpenoids (linalool and ocimene) as well as benzenoids

(methylbenzoate and benzyl acetate) and a phenylpropanoid (phenylethyl acetate). A few

other compounds were also detected at low level such as linalool oxide, phenylethanol,

nerolidol, indole and formanilide. Determination of the compounds was based on

similarity of the mass spectra and the spectrum fragments of the compounds with the

compounds in the NIST mass spectral library 2002 with the cut off value of Similarity

Index (SI) equal to or more than 90%. Floral scent analyses in a 24-hour cycle in Figures

6 and 7 show that the emission of major terpenoids compounds (ocimene, linalool) as

well as benzenoids (methylbenzoate and benzyl acetate) and the phenylpropanoid

(phenylethyl acetate) started as early as 6.00am, and increased gradually until it reached

the highest peak at 2.00 noon. After that, the emission of the compounds decreased

gradually until 6.00pm. None of the volatile compounds was detected from the flowers

by GC-MS at night (from 8.00pm until 4.00am). During the highest peak of the scent

Page 75: Vanda Mimi Palmer Thesis

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Figure 5: Gas Chromatogram of Volatile Compounds Emitted by Fully-open Flower of Vanda Mimi Palmer. The compounds

detected were: (1) ocimene, (2) linalool oxide, (3) linalool, (4) methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7) formanilide,

(8) phenylethyl acetate, (9) indole, and (10) nerolidol.

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Table 4: Volatile Compounds Emitted by Vanda Mimi Palmer with Their Relative

Retention Time and m/z Fragments

Peak Relative

retention time

(min)

Main spectrum fragments

(m/z)

Compound

name

Monoterpene

1

2

3

8.636

9.147

9.592

36,41,53,67,79,93,105,121

41,43,59,81,93,112

41,43,69,71, 93,107,121,136

Ocimene

Linalool oxide

Linalool

Sesquiterpene

10

16.492

41,43,69,71,93,107,123,136,162

Nerolidol

Benzenoid

4

5

9.702

10.030

51,77,105,136

39,51,65,78,91,105,122

Methylbenzoate

Benzyl acetate

Phenylpropanoid

6

8

10.783

11.926

39,43,65,79,91,108,150

39,43,65,78,91,104

Phenylethanol

Phenylethyl

acetate

Indole

9

13.084

39,50,63,74,90,117

Indole

Formanilide

7

12.260

39,52,65,76,93,161

Formanilide

Page 77: Vanda Mimi Palmer Thesis

77

Emission of Terpenoid Compounds in a 24-hour cycle

0

250000

500000

750000

1000000

1250000

1500000

1750000

2000000

2250000

2500000

12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Time

Ab

un

da

nc

e

ocimene

linalool

nerolidol

linalool oxide

Figure 6: Emission of Terpenoids by Fully-open Flower of Vanda Mimi Palmer in a

24-hour Cycle: Ocimene, Linalool, Nerolidol and Linalool Oxide. The volatile

compounds of fully-open flower of Vanda Mimi Palmer were captured by solid phase

micro-extraction (SPME) before injection into GC-MS injector port. The experiment was

carried out for 12 hours in light, followed by 12 hours in dark. The error bars represent

the standard deviation of the relative amount of the volatile compounds detected in three

replicates of scent analysis.

Page 78: Vanda Mimi Palmer Thesis

78

Emission of Benzenoid/Phenylpropanoid Compounds in a 24-hour cycle

0

250000

500000

750000

1000000

1250000

1500000

1750000

2000000

2250000

2500000

2750000

3000000

3250000

3500000

3750000

4000000

12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Time

Ab

un

da

nc

e

methylbenzoate

benzyl acetate

phenylethyl acetate

phenylethanol

Figure 7: Emission of Benzenoids and Phenylpropanoids of Vanda Mimi Palmer in

a 24-hour Cycle: Methylbenzoate, Benzyl Acetate, Phenylethyl Acetate and

Phenylethanol (Phenylethyl Alcohol). The volatile compounds of fully-open flower of

Vanda Mimi Palmer were captured by solid phase micro-extraction (SPME) before

injection into GC-MS injector port. The experiment was carried out for 12 hours in light,

followed by 12 hours in dark. The error bars represent the standard deviation of the

relative amount of the volatile compounds detected in three replicates of scent analysis.

Page 79: Vanda Mimi Palmer Thesis

79

emission of Vanda Mimi Palmer (at 2.00pm), the scent of Vanda Mimi Palmer was

represented by 34.8% of terpenoids, 62.6% of benzenoids and phenylpropanoids, and

2.6% traces of other compounds including indole and formanilide (Figure 8A). The major

terpenoids detected in the scent of Vanda Mimi Palmer which were ocimene and linalool,

representing 34% of the total scent (ocimene 16.3% and linalool 17.7%) (Figure 8B).

Another terpenoids including linalool oxide (monoterpene) and nerolidol (seisquiterpene)

were detected at trace levels during the highest peak of scent emission which represent

0.3% and 0.5%, respectively, of the total scent (see Figure 8B). While for benzenoids and

phenylpropanoids, phenylethyl acetate was detected at the highest level representing

26.4% of the total scent of Vanda Mimi Palmer, followed by benzyl acetate (23.7%),

methylbenzoate (11.1%) and phenylethyl alcohol (phenylethanol) (1.4%) (see Figure 8B).

Scent analyses of Vanda Mimi Palmer at three developmental stages (bud, half-open

flower, and fully-open flower) in Figure 9 show no detectable volatile compounds

emitted at bud stage. In the half-open flower stage, emission of terpenoids incuding

linalool and ocimene was detected at a very high level compared to other compounds. In

the fully-open flower stage, scent emission of Vanda Mimi Palmer is at the highest level.

In contrast to half-open flower stage, the benzenoid and phenylpropanoid compounds

including benzyl acetate and phenylethyl acetate were detected at the highest level from

fully-open flower stage.

Page 80: Vanda Mimi Palmer Thesis

80

benzenoids and

phenylpropanoids

62.6%

other compounds

2.6%

terpenoids

34.8%

ocimene

16.3%

linalool oxide

0.3%

linalool

17.7%

methylbenzoate

11.1%

phenylethyl alcohol

1.4%

formanilide

0.4%

benzyl acetate

23.7%

phenylethyl acetate

26.4%

indole

2.2%

nerolidol

0.5%

Figure 8: Percentage of (A) Terpenoid, Benzenoid and Phenylpropanoid, and other

Compounds and (B) Each Compound in the Scent of Fully-open Flower of Vanda

Mimi Palmer. The pie charts show the percentage of each individual compounds and

class of compounds detected in the scent of Vanda Mimi Palmer during the highest peak

of scent emission at 2.00pm.

(A)

(B)

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81

Figure 9: Comparison of Volatiles Emitted by Vanda Mimi Palmer at Three

Different Flower Developmental Stages: (A) bud (B) half-open flower and (C) fully-

open flower. The compounds are; (1) ocimene, (2) linalool oxide, (3) linalool, (4)

methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7) formanilide, (8) phenyletyl

acetate, (9) indole and (10) nerolidol.

Page 82: Vanda Mimi Palmer Thesis

82

The volatile compounds emitted by Vanda Mimi Palmer were compared with compounds

emitted by both parents, Vanda Tan Chay Yan (non-scented orchid) and Vanda tessellata

(scented orchid) in order to understand the fragrance characteristic of Vanda Mimi

Palmer. Emission of some volatile compounds including ocimene, decane and copaene in

Vanda Tan Chay Yan (Figure 10) were determined using GC-MS. The same GC-MS

analysis was not carried out for Vanda tessellata due to inavailability of the sample

source. Thus, a previously reported result by Kaiser (1993) on the scent of Vanda

tessellata was used for comparison. In his study, fully-open flower of Vanda tessellata

was trapped into a vessel for three hours (11.00 am-2.00 pm) and pumped into 10mg of

pure activated charcoal and then dissolved in 10-50µl of carbon disulphate followed by

10-30µl of ethanol solution before injecting into GC-MS (GC- Carlo Erba FTV 4160

chromatograph, MS- Varian MAT Model 212/CH-5 mass spectrometer with Finnigan

INCOS data system) (Kaiser, 1993). A comparison of the volatile compounds emitted by

Vanda Mimi Palmer and both parents is shown in Table 5. From the comparison,

ocimene was the only compound found to be emitted by Vanda Mimi Palmer and both of

its parents. Some of the compounds that were detected in the scent of Vanda Mimi

Palmer were also present in one of its parents (Vanda tessellata, a scented orchid) which

included benzyl acetate, methylbenzoate and linalool (Kaiser, 1993). Meanwhile, other

compounds emitted by Vanda Mimi Palmer included nerolidol, phenylethanol and

phenylethyl acetate were not detected in both parents.

Page 83: Vanda Mimi Palmer Thesis

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Figure 10: Comparison of Volatiles Emitted by Fully-open Flower of (A) Vanda Mimi Palmer and (B) Vanda Tan Chay Yan. The compounds are: (1) ocimene, (2) linalool oxide, (3) linalool, (4) methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7)

formanilide, (8) phenyletyl acetate, (9) indole and (10) nerolidol (11) decane and (12) copaene.

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Table 5: Comparison of Terpenoids, Benzenoids and Phenylpropanoids Emitted by

Vanda Mimi Palmer, Vanda tessellata and Vanda Tan Chay Yan

Compound Vanda Mimi

Palmer

Vanda Tan Chay

Yan

Vanda tessellata

(Kaiser, 1993)

Monoterpene

α-pinene

Linalool

Linalool oxide

Mycrene

Ocimene

Sesquiterpene

Copaene

Nerolidol

-

-

-

-

-

-

-

-

-

-

-

Benzenoid

Benzaldehyde

Benzyl acetate

Benzyl alcohol

Cinnamyl alcohol

Methyl benzoate

Methyl cinnamate

Methyl salicylate

Phenylpropanoid

Phenylethanol

Phenylethyl acetate

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Note: The “ √ ” indicates the presence of the compound in the volatiles of the flowers

while the absence of the compound is indicated by “ - ”.

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4.1.2 Analysis of Essential Oil of Vanda Mimi Palmer and Vanda Tan Chay Yan

Essential oil extraction was carried out on fully-open flower of Vanda Mimi Palmer and

Vanda Tan Chay Yan separately in order to understand the scent production and storage

in Vanda Mimi Palmer. The essential oil was extracted by soaking the flowers for 48

hours in hexane and followed by GC-MS analysis. The GC-MS detected 63 compounds

in the essential oil of Vanda Mimi Palmer and 63 compounds in the essential oil of Vanda

Tan Chay Yan (Appendix D, subsection (c) and (d)). Comparison between the

compounds detected in the essential oil of both flowers is presented in Table 6. Some of

the volatile compounds detected in the scent of Vanda Mimi Palmer (Figure 4 in section

4.1.1) which included linalool, methylbenzoate, phenylethanol, benzyl acetate and

phenylethyl acetate were also detected in the essential oil of Vanda Mimi Palmer (see

Table 6). Besides that, other intermediate compounds such as benzyl alcohol and benzyl

benzoate were also detected in the essential oil of Vanda Mimi Palmer. In addition,

another two sesquiterpenes which were germacrene and copaene were detected in the

essential oil of Vanda Mimi Palmer. Based on the comparison, phenylethanol compound

which was detected in the scent of Vanda Mimi Palmer., was also detected in the

essential oil of both Vanda Mimi Palmer and Vanda Tan Chay Yan. Interestingly, the

presence of phenylethanol compound in the essential oil of Vanda Mimi Palmer was

detected at high level representing 11.18% of the total essential oil (see Table 6). The

essential oil analysis shows that the terpenoids represent 2.09% of while benzenoids and

phenylpropanoids represent 26.51% of the total essential oil (Figure 11).

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Table 6: Comparison of Terpenoids, Benzenoids and Phenylpropanoids Detected in

Essential Oils of Vanda Mimi Palmer and Vanda Tan Chay Yan

Compound

Vanda Mimi Palmer

Vanda Tan Chay Yan

Monoterpene

1-hydroxylinalool

Linalool

Sesquiterpene

Copaene

germacrene D

-

√ (1.79 %)

√ (0.17 %)

√ (0.13 %)

√ (0.38 %)

-

-

-

Benzenoid

Benzylacetaldehyde

Benzyl acetate

Benzyl alcohol

Benzyl benzoate

Methylbenzoate

Naphthalene

Phenylpropanoid

Phenylethanol

Phenylethyl acetate

Vanillin

√ (1.55 %)

√ (2.02 %)

√ (4.98 %)

√ (0.20 %)

√ (3.37 %)

√ (0.15 %)

√ (11.18 %)

√ (0.85 %)

√ (2.21 %)

√ (3.57 %)

-

-

-

-

-

√ (2.00 %)

-

-

Note: The “ √ ” indicates the presence of the compound in the essential oil of the flowers

while the absence of the compound is indicated by “ - ”. The full list and percentage of

volatiles, semi-volatiles and non-volatiles in the essential oil of both Vanda Mimi Palmer

and Vanda Tan Chay Yan can be referred to Appendix D, in subsection (c) and (d).

Page 87: Vanda Mimi Palmer Thesis

87

terpenoids

2.09%

benzenoids and

phenylpropanoids

26.51%

other compounds

71.40%

Figure 11: Composition of Terpenoids, Benzenoids and Phenylpropanoids, and

Other Compounds in the Essential Oil of Vanda Mimi Palmer.

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4.2 Molecular Studies of Fragrance-related cDNA Transcripts of Vanda Mimi

Palmer

4.2.1 Isolation of Putative Fragrance-related cDNAs

4.2.1.1 Floral cDNA Library Screening

Isolation of fragrance-related cDNAs from Vanda Mimi Palmer was carried out by

screening the floral cDNA library representing all mRNA transcripts expressed in a 24-

hour cycle (12 hours in light and 12 hours in dark) and at different developmental stages

including bud, half-open flower, and fully-open flower. In a primary screening, 500,000

phages (500,000 pfu) were screened by hybridizing the floral cDNA library with fully-

open flower cDNA probe of Vanda Mimi Palmer representing all mRNA transcripts

expressed in fully-open flower between 8.00am to 6.00pm, a period when strong scent

emission of Vanda Mimi Palmer was detected (refer Figures 6 and 7). From the primary

screening (Figure 12a), 800 plaques showed positive signals on X-ray film by

autoradiography detection method. The plaques with positive signals represent mRNA

transcripts that are expressed between 8.00am to 6.00pm in fully-open flower of Vanda

Mimi Palmer. Secondary screening was applied to all the putative positive plaques

(Figure 12b) and the plaques with the strongest signal on X-ray film were cored out and

selected for in vivo excision.

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Figure 12: Autoradiograph Containing Positive Signals (dark spot) of Putative

Positive Plaques from (a) Primary Screening; (b) Secondary Screening. From the

secondary screening, individual plaques with the strongest signal were cored out for in

vivo excision.

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4.2.1.2 Reverse-Northern Analysis

All the clones were in vivo excised to phagemid form and used for reverse-Northern

analysis. The reverse-Northern analysis was carried out by hybridizing PCR-amplified

inserts (Figure 13a) with two different cDNA probes synthesized from mRNA transcripts

of fully-open flower (scent emitting stage) and bud (non-scent emitting stage) of Vanda

Mimi Palmer separately in order to isolate the clones that putatively represent fragrance-

related transcripts. From a total of 800 putative positive clones, 246 clones showed up-

regulated expression in fully-open flower stage during day time compared to the bud

stage (Figures 13b and 13c). A previous study on floral cDNA library of Vanda Mimi

Palmer has shown that half of the library was contaminated by transcripts encoding

Cymbidium mosaic virus coat protein (Chan, 2009). Thus, clones containing Cymbidium

mosaic virus coat protein transcripts (Figure 13d) were removed by hybridizing the PCR-

amplified cDNA inserts of the clones with probes representing the Cymbidium mosaic

virus coat protein transcripts. Upon eliminating transcripts related to Cymbidium mosaic

virus, only 62 clones were selected for plasmid isolation and sequenced as candidates for

fragrance-related cDNAs.

4.2.1.3 Sequencing and Analysis

Single pass sequencing result of the 62 clones were assembled into 5 contigs and 52

singletons using the Cap3 sequence assembly program (Appendix E). All of the 62

sequences (100%) were readable sequences with more than 285bp. The tentative unique

Page 91: Vanda Mimi Palmer Thesis

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Figure 13: Hybridization of (a) PCR Amplified Inserts with Three Different Probes;

(b) Fully-open Flower cDNA Probe, (c) Bud Stage cDNA Probe, and (d) Cymbidium

mosaic Virus cDNA Probe. White-circles show selected clones that have up-regulated

expression in fully-open flower of Vanda Mimi Palmer during day time compared to bud

stage after removal of clones containing Cymbidium mosaic virus coat protein cDNA.

Lane 1-25 represents PCR product of putative positive clones isolated from the screening

of floral cDNA library.

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92

gene sequences (TUG) were aligned against the GeneBank nucleotide and protein

database (BLASTX) to search for sequence similarity with the cut off value less than 1e-

05 and score more than 80. From the BLASTX search, fifty-seven tentative unique gene

sequences (TUG) were similar to the sequences in NCBI database (Appendix E). The

other five sequences had e-value more than 1e-05

and were classified as sequences with

no-significant hit due to no similarity sequence in the NCBI database. All the ESTs were

classified into eleven groups based on their putative functionality (Zhao et al., 2006). The

groups are metabolism (19%), unknown (16%), protein synthesis (11%), cellular

transport (10%), cell cycle and DNA processing (10%), protein with binding function

(8%), energy (8%), cell rescue, defense and virulence (6%), development (6%),

biogenesis of cellular component (3%) and transcription (3%) (see Figure 14 and

Appendix E). The unknown group represents ESTs with unknown function including the

sequences that hit to hypothetical proteins and also with non-significant hit sequences.

4.2.1.4 Verification of Putative Fragrance-related cDNAs with Up-regulated

Expression in Fully-open Flower Compared to Bud of Vanda Mimi Palmer

Based on BLASTX and literature search results, it was postulated that some cDNA

transcripts classified in metabolism and unknown group might be potential putative

fragrance-related cDNAs. Thus, a verification was carried out for thirteen selected clones

(potential putative fragrance-related cDNA clones) by RT-PCR using two different

Page 93: Vanda Mimi Palmer Thesis

93

metabolism

19%

unknown

16%

protein synthesis

11%cellular transport

10%

cell cycle and DNA

processing

10%

protein with binding

function

8%

energy

8%

Cell rescue, defence

and virulence

6%

Development

6%

biogenesis of cellular

component

3%

transcription

3%

Figure 14: Classification of the Clones with Up-regulated Expression in Fully-open

Flower Compared to Bud of Vanda Mimi Palmer. The 62 clones with up-regulated

expression in fully-open flower compared to bud of Vanda Mimi Palmer were classified

into eleven groups based on their putative functionalities.

Page 94: Vanda Mimi Palmer Thesis

94

tissues which are fully-open flower and bud to choose for the cDNAs clones that have

up- regulated expression in the fully-open flower. Three cDNA clones from the

metabolism group including putative esterase (VMPEST), putative 4-(cytidine 5′-

diphospho)-2-C-methyl-d-erythritol kinase (VMPCMEK) and putative cytochrome p450

protein (VMPCyP450) were selected for the verification because the selected transcripts

have been reported to be involved in fragrance biosynthesis in other plants. Besides that,

another 10 putative fragrance-related transcripts from unknown group including

hypothetical protein and no significant hit protein transcripts were selected for

verification of their expression levels in the two different tissues. The cDNAs from this

group could be novel transcripts involved in the fragrance biosynthetic pathway.

From the verification results, 5 clones showed up-regulated expression in fully-open

flower compared to bud, 3 clones showed down-regulated expression in fully-open

flower compared to bud and 6 clones showed equal expression in both fully-open flower

and bud stages (see Table 7). Three putative fragrance-related cDNA clones which

showed up-regulated expression in fully-open flower compared to bud of Vanda Mimi

Palmer were selected for full-length cDNA isolation and expression analysis by real-time

RT-PCR. The selected cDNA clones were putative 4-(cytidine 5′-diphospho)-2-C-

methyl-d-erythritol kinase (hereafter referred as VMPCMEK), putative cytochrome p450

protein (hereafter referred as VMPCyP450) and an unknown protein cDNA transcript

(hereafter referred as VMPA28).

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Table 7: Verification of Putative Fragrance-related cDNAs with Up-regulated

Expression in Fully-open Flower Compared to Bud of Vanda Mimi Palmer by RT-

PCR; (a) cDNA transcripts of the selected clones were PCR-amplified using first-strand

cDNA of the selected tissues. (b) The PCR products were hybridized with specific probe

representing the cDNA sequence of each transcript (reverse Northern). Each lane

representing different tissues selected for the verification; 1) bud of Vanda Mimi Palmer

2) fully-open flower of Vanda Mimi Palmer. “No significant hit” and “hypothetical

protein” transcript sequences were named as unknown protein.

Clone Name PCR and reverse Northern Housekeeping gene

(elongation factor)

51 Putative cytochrome

P450 (VMPCyP450)

(up-regulated in fully-

open flower stage)

1 2

1 2

31 Putative esterase

(VMPEST)

(down-regulated in fully-

open flower stage)

1 2

1 2

71 Putative 4-(cytidine 5'-

diphospho)-2-C-methyl-

D-erythritol kinase

(VMPCMEK)

(up-regulated in fully-

open flower stage)

1 2

1 2

33 Hypothetical protein

(up-regulated in fully-

open flower stage)

1 2

1 2

A36 Unknown protein

(equal expression)

1 2

1 2

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a) (b)

(a)

(b)

(a)

(b)

(a)

(b)

Page 96: Vanda Mimi Palmer Thesis

96

Clone Name PCR and reverse Northern Housekeeping gene

(elongation factor)

48 Hypothetical protein

(up-regulated in fully-open

flower stage)

1 2

1 2

59 Unknown protein

(down-regulated in fully-

open flower)

1 2

1 2

83 Hypothetical protein

(equal expression)

1 2

1 2

90 Hypothetical protein

( down-regulated in fully-

open flower stage )

1 2

1 2

96 Unknown protein

(down-regulated in fully-

open flower)

1 2

1 2

A28 Unknown protein

(up-regulated in fully-open

flower)

1 2

1 2

A46 Unknown protein

(equal expression)

1 2

1 2

A54 Hypothetical protein

(equal expression)

1 2

1 2

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

(a)

(b)

Page 97: Vanda Mimi Palmer Thesis

97

4.2.2 Cloning and Characterization of Selected Fragrance-related Transcripts

Molecular characterization was carried out on three putative fragrance-related transcripts

that showed up-regulated expression in fully-open flowers of Vanda Mimi Palmer. The

selected transcripts were putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol

kinase (VMPCMEK), putative cytochrome P450 protein (VMPCyP450) and an unknown

protein (VMPA28). In addition, a putative phenylacetaldehyde synthase (VMPPAAS)

previously identified from the floral ESTs of Vanda Mimi Palmer by Miss Chan Wai Sun

was also selected for molecular characterization. Putative fragrance-related cDNA

transcripts were subjected to full-length cDNA isolation and gene expression analysis.

Gene expression analysis was carried out in different tissues, at different developmental

stages, and different time points in a 24-hour cycle.

4.2.2.1 Sequence and Expression Analyses of Putative Phenylacetaldehyde Synthase

(VMPPAAS)

The full-length sequence of VMPPAAS cDNA is 1709 bp consisting of 1524bp open

reading frame (ORF) flanked by a 92bp 5‟ untranslated region (UTR) and a 93bp 3‟UTR

including a poly-A tail (Figure 15). The ORF encodes a protein of 508 amino acid

residues (Figure 16). The predicted molecular weight of this protein is 56.1 kD with an

isoelectric point (pI) of 7.1.

Page 98: Vanda Mimi Palmer Thesis

98

3 GCG GGA TCG CGG CAA GGC AGA AGG CAA CCA AAA AAC AAA AAA AAA 47

48 AAA AAC AAG CTT GTT GCC TTT CCC ATT CCC TAG GAA CTC CAG AAA 92

>>>>>>>>>>>>>>>

93 ATG GGC AGC CTT CCC ACC GAA CCA TTC CTG CCA CTA GAC CCA GAC 137

1 M G S L P T E P F L P L D P D 15

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

VMPPAAS ORF Forward primer

138 CGC TTC ACC AAA GAA TCC AAA GCC GTC GTC GAC TTC ATC GCC GAC 182

16 R F T K E S K A V V D F I A D 30

183 TAC TAC CGC CAA ATA GAA CTC TTC CCT GTT CGC AGC CAA GTA AAG 227

31 Y Y R Q I E L F P V R S Q V K 45

228 CCA GGC TAT CTC CAT GAC CGC ATT CCA AAC ACT CCC CCC ATC CTC 272

46 P G Y L H D R I P N T P P I L 60

273 TCC GAA CCC ATC ACC ACA ATC CTC CAC GAC ATT AAA ACA GAC ATC 317

61 S E P I T T I L H D I K T D I 75

318 TTT CCC GGA CTA ACC CAC TGG CAA AGC CCC AAT TTT TAC GGC TAC 362

76 F P G L T H W Q S P N F Y G Y 90

363 TAC CAA GCC AAT GCC AGC ACC CCC GGT TTC GCC GGA GAG ATG CTC 407

91 Y Q A N A S T P G F A G E M L 105

408 TGT TCC GGC CTC AAC GTC GTC GGC TTC AGC TGG ATC GCT TCC CCT 452

106 C S G L N V V G F S W I A S P 120

453 GCC GCC ACT GAA CTA GAA ACC ATC ATC ATG GAC TGG ATG GCC AAG 497

121 A A T E L E T I I M D W M A K 135

498 ATG CTC AAA CTT CCA TCA ACC TTC CTT TCC GGA CAC CTC GGC GGC 542

136 M L K L P S T F L S G H L G G 150

543 GGC GGT GGC GTA ATC CAC GGC AGC ACG TGC GAA GCG GTG CTC TGC 587

151 G G G V I H G S T C E A V L C 165

588 ACC CTC GCC GCT GCT AGA GAT AAC GCT TTG AGC AAG AGC GAC GGC 632

166 T L A A A R D N A L S K S D G 180

633 GAA GGG ATC ACG AAG CTG ACG GTA TAT GTC TCT GAT CAG ACA CAT 677

181 E G I T K L T V Y V S D Q T H 195

678 TTT ACG GTT CAG AAG GCG GCG AAG TTG GTT GGA ATC CCG ACG CGG 722

196 F T V Q K A A K L V G I P T R 210

723 AAC TTA CGG GTG ATA TCG ACT TCG AGG GAG ACA GGG TAT GCC TTG 767

211 N L R V I S T S R E T G Y A L 225

768 ACG GCG GAG ATT GTG AGG GCG GCG ATG GAT GCT GAT GTG GCG GCA 812

226 T A E I V R A A M D A D V A A 240

813 GGG ATG GTG CCG CTG TAT TTG TGT GGC ACG GTG GGG ACG ACG GCT 857

241 G M V P L Y L C G T V G T T A 255

858 GTG GGG GCG GTG GAC CCG ATA AGG GAG ATC GGG GAG GTT GCG AGG 902

256 V G A V D P I R E I G E V A R 270

903 GAG TTC GGG GTG TGG TTC CAC GTG GAC GCG GCG TAT GCG GGG AGC 947

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271 E F G V W F H V D A A Y A G S 285

948 GCT GGG ATT TGC CCT GAG TTC CGG CGG TTC TTT GAT GGA GTG GAG 992

286 A G I C P E F R R F F D G V E 300

993 ACG GCT GAT TCC TTT AGT TTG AAT CCG CAT AAA TGG CTG CTC GCA 1037

301 T A D S F S L N P H K W L L A 315

1038 AAC ATG GAC TGT TGT TGC CTT TGG GTA AGA TGT GCA ACG AAG CTC 1082

316 N M D C C C L W V R C A T K L 330

1083 GTA GAC TCG TTA TCG ACC AAG CCG GAG ATA TTG ACA AAC AGT GCT 1127

331 V D S L S T K P E I L T N S A 345

1128 AGC GAA GAT GGC AAA GTG ATT GAC TAC AAA GAT TGG CAG GTC GCA 1172

346 S E D G K V I D Y K D W Q V A 360

1173 CTG AGT CGT AGG TTT CGT GCA ATG AAG CTA TGG ATA GTC ATC AGA 1217

361 L S R R F R A M K L W I V I R 375

1218 CGA TTT GGA GTT GCT AAC CTG ATG GAG CAC ATC AGG AGC GAT GTG 1262

376 R F G V A N L M E H I R S D V 390

1263 GAG ATG GCC AAG CAT TTC GAG AGA CTT GTC GCC GAG GAT GAG AGG 1307

391 E M A K H F E R L V A E D E R 405

1308 TTT GAG GTG GTT GTA CCA AGA AGA TTC ACG CTC GTT TGT TTT AAA 1352

406 F E V V V P R R F T L V C F K 420

1353 TTG AGG TAT GTG GGA GAA GAT ATT GAT GAA GAG GAG GGG ACG AAA 1397

421 L R Y V G E D I D E E E G T K 435

>>>>>>>

1398 TGT TGG GAG ATG AAT AAG AAG TTG CTC GAT TCG GTG AAC GAA AGT 1442

436 C W E M N K K L L D S V N E S 450

>>>>>>>>>>>>>>>>>>>>>

VMPPAAS RT-PCR Forward Primer

1443 GGA CGA GCA TTC ATG ACC CAT GCG GTT GTT TGC GGG CAG TTT GTG 1487

451 G R A F M T H A V V C G Q F V 465

1488 CTG CGG TTT GCA CTT GGC GCC ACG TTG ACA GAG ATA CGA CAT GTG 1532

466 L R F A L G A T L T E I R H V 480

1533 GAG GAG ACA TGG AGG TTG GTT CAA GAG AAG GCA AGT GAG TTG TTG 1577

481 E E T W R L V Q E K A S E L L 495

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

VMPPAAS RT-PCR Reverse Primer

1578 ATG ATT ACG AAT GAG CTG GGT TGG AAA CTC AAA ACA CTC TGA GAT 1622

496 M I T N E L G W K L K T L * 508

<<< <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

VMPPAAS ORF reverse primer

1623 AGC TCA CTT AAA ATA AAA AGA GAA ATA ATA GTT TCA AAT GAA ATA 1667

1668 AAA TTA TAA AAA ATT TAA AAA TTT AAA GAA AAA AAA AAA AAA 1709

Figure 15: The Nucleotide and Deduced Amino Acid Sequence of VMPPAAS

(Putative Phenylacetaldehyde Synthase). The open reading frame (ORF) of

VMPPAAS starts at nucleotide 93. The asterisk (*) indicates stop codon. The putative

polyadenylation signal is bold and underline. The location of gene specific primers used

in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).

Page 100: Vanda Mimi Palmer Thesis

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Figure 16: PCR Product of the Open Reading Frame (ORF) of Putative

Phenylacetaldehyde Synthase of Vanda Mimi Palmer (VMPPAAS). The ORF

represents 508 amino acids. The PCR product was electrophoresed on 1% (w/v) agarose

gel. Lane M: 100bp marker (Vivantis, Malaysia); lane 1 and 2: gap; lane 3: PCR product

of ORF of VMPPAAS.

1500bp

1000bp

500bp

M 1 2 3

~1550bp 2000bp

3000bp

Page 101: Vanda Mimi Palmer Thesis

101

BLASTX and BLASTP analysis showed that VMPPAAS has the closest similarity to

putative tyrosine/dopa decarboxylase (AAK50420.1) and pyridoxal-dependent

decarboxylase conserved domain containing protein (ABB47543.1) of Oryza sativa from

japonica-cultivar group, corresponding to 57% identity. The VMPPAAS also shows

similarity to tyrosine decarboxylase of Aristolochia contorta (ABJ16446.1) and

tyrosine/dopa decarboxylase of Papaver somniferum (AAC61844.1) corresponding to

55% and 54% identities, respectively. Besides that, VMPPAAS also shows similarity to

fragrance-related amino acid sequences of well studied scented flowers including

aromatic L-amino acid decarboxylase of Rosa hybrida (BAF64844.1) (56% identity),

phenylacetaldehyde synthase of Rosa hybrida (ABB04522.1) (56% identity) and

phenylacetaldehyde synthase of Petunia hybrida (ABB72475.1) (54% identity).

Localizome analysis using Localizome software (Lee et al., 2006) revealed a domain for

pyridoxal-dependent decarboxylase in the amino acid sequence of VMPPAAS. In

addition, the same software also predicted no signal peptide present in the amino acid

sequence of VMPPAAS and in all of the closely related protein sequences. Prediction of

motifs present in the deduced protein sequence of VMPPAAS and the closely related

proteins by the Expasy tool showed a conserved motif for a pyridoxal phosphate

attachment site (see Figure 17). Besides that, there are two N-myristoylation sites

(GVxxGS and GAxxTE), three casein kinase II phosphorylation sites (TxxE, SxxE, and

TxxE), two N-glycosylation sites (NAST and NESG), and a site for cAMP- and c-GMP-

dependent protein kinase phosphorylation (RRxT) in the VMPPAAS amino acid

sequence (see Figure 17). There is another motif (VHVDAAY) shared by VMPPAAS

Page 102: Vanda Mimi Palmer Thesis

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Figure 17: Alignment of VMPPAAS (Putative Phenylacetaldehyde Synthase) with

Other Closely Related Protein Sequences from the GeneBank. The VMPPAAS amino

acids sequence was aligned with putative tyrosine/dopa decarboxylase (AAK50420.1) of

Oryza sativa, tyrosine/dopa decarboxylase of Papaver somniferum (AAC61844.1),

aromatic L-amino acid decarboxylase of Rosa damascena (BAF64843.1),

phenylacetaldehyde synthase of Rosa hybrida (ABB04522.1) and phenylacetaldehyde

synthase of Petunia hybrida (ABB72475.1). The pyridoxal phosphate attachment site and

HVDAAY motif are highlighted in box. Four stars (****) indicate the conserved motif

for N-myristoylation sites (GVxxGS and GAxxTE) meanwhile two stars (**) indicate the

conserved motif for casein kinase II phosphorylation sites (TxxE, SxxE and TxxE). The

conserved motif for N-glycosylation sites (NAST and NESG) are marked with three stars

(***).

VHVDAAY motif

Pyridoxal phosphate attachment site

NAST

***

TxxE

***

GVxxGS

****

SxxE

**

TxxE

**

NESG

***

Page 103: Vanda Mimi Palmer Thesis

103

and other decarboxylases protein either from plants or bacteria (Connil et al., 2002) (see

Figure 17). A phylogenetic tree (Figure 18) was constructed using MEGA version 4.0

(Tamura et al., 2007) to estimate the genetic relatedness between amino acid sequence of

VMPPAAS with other plant decarboxylases. In the phylogenetic tree, VMPPAAS is

clustered together with protein sequences from aromatic amino acid of Ricinnus

communis, and supported by the bootstap value of 93 for the branch.

Relative expression study of VMPPAAS transcript in different tissues (Figure 19) of

Vanda Mimi Palmer showed up-regulated expression in floral tissues including petal,

sepal and lip but down-regulation in vegetative tissues including leaf, shoot, root and

stalk. Among the floral tissues, petal has shown the highest expression of VMPPAAS

transcript which is more than 20,000-fold expression compared to bud tissue as the

calibrator. In the sepal, the expression of VMPPAAS transcript was also detected at high

level (nearly to 15,000-fold expression) compared to the calibrator. Meanwhile in the lip,

the VMPPAAS transcript was detected at 5000-fold higher expression compared to the

calibrator.

Relative expression analysis at five different flower developmental stages shows that the

VMPPAAS transcript was differentially expressed during the flower life cycle (Figure

20). In bud stages including young and mature buds, the expression of VMPPAAS

transcript was down-regulated compared to half-open and fully-open flower stages. At

14-day fully-open flower, the expression of VMPPAAS transcript decreased drastically at

very low level.

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VMPPAAS (Vanda Mimi Palmer)

OsTyDC(Oryza sativa-japonica cultivar...

RcAADC (Ricinus communis)

PsTyDC(Papaver somniferum)

RsAADC (Rosa hybrida)

RhPAAS (Rosa hybrida)

RdAADC (Rosa x damascena)

PhPAAS (Petunia hybrida)

CaTDC(Camptotheca acuminata)

AtTyDC (Arabidopsis thaliana)

ZmTyDC1 (Zea mays)

OsTDC (Oryza sativa Japonica Group)

ZmTyDC2 (Zea mays)

Figure 18: Phylogenetic Tree of VMPPAAS with Homologous Proteins. The

phylogenetic tree is constructed by MEGA software version 4.0 using amino acid

sequence of VMPPAAS with the homologous proteins available in GeneBank database

(NCBI) including tyrosine decarboxylase of Oryza sativa (AAK50420.1), aromatic

amino acid decarboxylase of Ricinus communis (EEF36965.1), tyrosine/DOPA

decarboxylases Papaver somniferum (AAC61844.1), aromatic L-amino acid

decarboxylase of Rosa hybrida (BAF64844.1), phenylacetaldehyde synthase of Rosa

hybrida (ABB04522.1), aromatic L-amino acid decarboxylase of Rosa damascene

(BAF64843.1), phenylacetaldehyde synthase of Petunia hybrida (ABB72475.1),

tryptophan decarboxylases of Camptotheca acuminata (AAB39708.1), tyrosine

decarboxylase of Arabidopsis thaliana (AAL69507.1), tyrosine/DOPA decarboxylase 1

of Zea mays (ACG29316.1), tryptophan decarboxylase of Oryza sativa (BAD35168.1)

and tyrosine/dopa decarbxylase 2 of Zea mays (ACG46884.1).

Page 105: Vanda Mimi Palmer Thesis

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0

5000

10000

15000

20000

25000

bud fully-

open

flower

petal sepal lip leaf root shoot stalk

Tissues

Rel

ativ

e

Exp

ress

ion

of

VM

PP

AA

S

(fo

ld c

han

ge)

Figure 19: Relative Expression Study of VMPPAAS Transcript in Different Tissues

of Vanda Mimi Palmer. The floral tissues are bud, fully-open flower, petal, sepal and lip

meanwhile the vegetative tissues are leaf, root, shoot and stalk. The quantitative

expression level of VMPPAAS in each tissue was calculated relative to the calibrator

which was the bud collected at 12.00 noon. Standard error between three replicates of

relative gene expression in each tissue is indicated by error bar.

0

15000

30000

45000

60000

75000

90000

105000

young bud

(green)

mature bud

(red)

half-open

flower

fully-open

flower

14-day old

flower

Developmental Stages

Rela

tive

Exp

ress

ion

of V

MPP

AAS

(fol

d ch

ange

)

Figure 20: Relative Expression Analysis on VMPPAAS Transcript at Different

Developmental Stages During Flower Development. This study was carried out on

five flower developmental stages of Vanda Mimi Palmer including young bud, mature

bud, half-open flower, fully-open flower and 14-day old fully-open flower. Quantitative

measurement of VMPPAAS expression in each flower developmental stage was

expressed relative to the calibrator which was the young bud collected at 12.00 noon.

Standard error between three replicates of relative gene expression at each developmental

stage is indicated by error bar.

Page 106: Vanda Mimi Palmer Thesis

106

Relative expression study of VMPPAAS transcript at different time points in a 24-hour

cycle (12 hours in light, followed by 12 hours in dark) (Figure 21) shows a differential

expression. From the analysis, the expression of VMPPAAS was higher at night (7.00pm

to 7.00 am) compared to day time (7.00am to 7.00pm). From 8.00am until 6.00pm (in

light) the expression level was lower compared to night. However, after 6.00pm the

amount of VMPPAAS transcript increased more than three fold with the highest peak at

10.00pm.

4.2.2.2 Sequence and Expression Analyses of Putative 4-(cytidine 5'-diphospho)-2-C-

methyl-D-erythritol Kinase (VMPCMEK)

The full-length cDNA sequence of VMPCMEK is 1446bp, comprising 1200bp ORF,

51bp of 5‟ UTR and 195bp of 3‟UTR with a polyadenylation tail (poly-A tail) (Figure

22). The ORF encodes a protein of 400 amino acid residues (Figure 23) with a predicted

molecular weight of 44.1 kD and an isoelectric point (pI) of 8.4.

BLASTX and BLASTP analysis (NCBI) shows the deduced amino acid sequence of

VMPCMEK exhibited similarity to 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol

kinase from other plants (66-73% identity). It has the highest similarity to 4-(cytidine 5'-

diphospho)-2-C-methyl-D-erythritol kinase of Nicotiana benthamiana (ABO87658.1),

Page 107: Vanda Mimi Palmer Thesis

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0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Time

Rela

tive e

xp

ressio

n o

f V

MP

PA

AS

(fo

ld c

han

ge)

Figure 21: Relative Expression Analysis of VMPPAAS at Different Time Points in a

24-hour Cycle. Quantitative expression of VMPPAAS at each time point was expressed

relative to the calibrator which was the fully-open flower collected at 12.00 noon.

Standard error between three replicates of relative gene expression at each time point is

indicated by error bar.

Page 108: Vanda Mimi Palmer Thesis

108

1 GGC TGC TGC AAA AAG CAT AAC TCC CTA CGC TTC TCA GCT TTT CTC 45

>>>>>>>>>>>>>>>>>>>>>>>

46 CTA ACA ATG GCC TCT TTC TCT AAT CAT CTG ATT TCA TCG TTC TCA 90

M A S F S N H L I S S F S 13

>>>>>>>>>>>>>>>>>

VMPCMEK ORF Forward Primer

91 TGC TCG AGA AGA AGT GCT TCA CTG CCC CGA AGA GGG AAT ACC CCC 135

14 C S R R S A S L P R R G N T P 28

136 TCG ATC TTT CGA CAT GGT CAT TAC TCT TTC AAA TCT TGG TCT GAG 180

29 S I F R H G H Y S F K S W S E 43

181 GTC AGC GGG AAC AAG TAT GGT AGA ATC CTC ATT TGT GCG GCA GAA 225

44 V S G N K Y G R I L I C A A E 58

226 ACT GGG AGG AGG CAA GTG GAG ATT GTT TAT GAT CCG GAG GAG AGG 270

59 T G R R Q V E I V Y D P E E R 73

271 TTT AGT GGA CTG GAG GGT GAA GTA GAT GAT AAC AAC AAG CTT TCT 315

74 F S G L E G E V D D N N K L S 88

316 AGG TTG ACC CTA TTC TCG CCG TGT AAG ATT AAT GTT TTC TTG AGG 360

89 R L T L F S P C K I N V F L R 103

361 ATA ACT GGA AAG AGG AAT GAT GGG TTT CAT GAT TTG GCC TCT TTG 405

104 I T G K R N D G F H D L A S L 118

406 TTT CAT GTA ATC AGT TTA GGA GAT ACG ATT AAA TTC TCC TTG TCA 450

119 F H V I S L G D T I K F S L S 133

451 CCA TTA AAG AGA AAG GAT CGC CTG TCA ACT AAT GTG CCG GGA GTT 495

134 P L K R K D R L S T N V P G V 148

496 CCA GTT GAT GAT AGA AAT TTG ATA ATC AGA GCT CTC AAT CTT TAC 540

149 P V D D R N L I I R A L N L Y 163

541 AGG AAG AAG ACA GGC ACA AAC AAT TTC TTC CAG ATT GAG CTT GAC 585

164 R K K T G T N N F F Q I E L D 178

586 AAA AAA GTT CCT ACT GGT GCT GGG CTT GGT GGT GGA AGT AGT AAC 630

179 K K V P T G A G L G G G S S N 193

631 GCA GCA ACT GCT TTA TGG GCT GCC AAC CAG TTC AGT CGT TCT CTT 675

194 A A T A L W A A N Q F S R S L 208

676 GTT ACT GAA AAA GAG CTT CAG GAT TGG TCA GGT GAA ATT GGT TCA 720

209 V T E K E L Q D W S G E I G S 223

721 GAT ATT CCT TTT TTT TTC TCT AAT GGG GCT GCA TAT TGT ACC GGT 765

224 D I P F F F S N G A A Y C T G 238

766 AGG GGA GAG GTT GTT AAA GAA CTT CCT TTT GCA TTG CCC AAG GAC 810

239 R G E V V K E L P F A L P K D 253

811 CTG CCA ATG GTT CTT ATA AAG CCC CAA GAA GCA TGT CCA ACC GCC 855

254 L P M V L I K P Q E A C P T A 268

856 GAA GTG TAC AAG CGA CTT CAT CTT GGT AAA ACT AGT TCA GTT GAC 900

269 E V Y K R L H L G K T S S V D 283

901 CCG TTG ACT CTG CTA GAA AAG ATA TCT CTA AAT GGA ATA TCT CAA 945

Page 109: Vanda Mimi Palmer Thesis

109

284 P L T L L E K I S L N G I S Q 298

946 GAT GTC TGC ATA AAT GAT CTT GAA CCC CCT GCA TTT GAT GTT TTG 990

299 D V C I N D L E P P A F D V L 313

991 CCA TCC TTG AAG AAG TTG AAG CAA CGT GTG CTA GCT GCA GGG CGT 1035

314 P S L K K L K Q R V L A A G R 328

1036 GGC CAG TAT AGT GCT GTT TTC ATG TCT GGA AGC GGA AGC ACC ATT 1080

329 G Q Y S A V F M S G S G S T I 343

>>>>>>>>>>>>>>>>>>>>>>>>>>>

VMPCMEK RT-PCR Forward Primer

1081 GTG GGA ATT GGT TCA CCA GAC CCA CCT CAA CTT GTT TAT GAT GAG 1125

344 V G I G S P D P P Q L V Y D E 358

1126 GAT GAA TAC AAT GAT GTT TTC ATA ACA GAG GCT TCC TTT CTC ACT 1170

359 D E Y N D V F I T E A S F L T 373

1171 CGG CAA CAG AAT CAG TGG TAC GCA GAG CCA ACT TCG TCC ACA GGG 1215

374 R Q Q N Q W Y A E P T S S T G 388

1216 TCT TTG AGC AGA GAA GAG CCG TCA CAA ACA GGA AAA TAA TTA CGA 1260

389 S L S R E E P S Q T G K * 400

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

VMPCMEK ORF Reverse Primer

<<<<<<<<<<<<<<<<<<<<<<<<<<

VMPCMEK RT-PCR Reverse Primer

1261 TAA TTT TTT TAC ATT CTA GAC CTT CTA ATT TTA ATT TTT CTC ACA 1305

1306 TAA AAT CAT ATT GTA TTA CTG TAC TTA TTG TTC ATG CAA GAA AGA 1350

1351 TCG ATC AAG CTA TCT TTC ATG AAT GAG CAA AAT ATG CAA TTT TAA 1395

1396 AAG GCA CAT TTA CAT GCT TAA AAA AAA AAA AAA AAA AAA AAA AAA 1440

1441 AAA AAA 1446

Figure 22: The Nucleotide and Deduced Amino Acid Sequences of VMPCMEK

(putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase). The open

reading frame (ORF) of VMPCMEK starts at nucleotide 52. The asterisk (*) indicates

stop codon. The putative polyadenylation signal in the nucleotide sequence is bold and

underline. The location of gene specific primers used in real-time PCR analysis and ORF

isolation are shown by arrow heads (>>>> and <<<<).

Page 110: Vanda Mimi Palmer Thesis

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Figure 23: PCR Product of the Open Reading Frame (ORF) of Putative 4-(cytidine

5'-diphospho)-2-C-methyl-D-erythritol Kinase of Vanda Mimi Palmer

(VMPCMEK). The PCR product was electrophoresed on 1% (w/v) agarose gel. Lane M:

100bp marker (Vivantis); Lane 1: PCR product of ORF of VMPCMEK.

1200bp

1000bp

500bp

M 1

~1200bp

Page 111: Vanda Mimi Palmer Thesis

111

corresponding to 73% identity. The closest similarity was followed by 4-(cytidine 5'-

diphospho)-2-C-methyl-D-erythritol kinase of Solanum lycopersicum (AAF87717.1) and

Catharanthus roseus (ABI35992.1), corresponding to 72% identity. Besides that, the

VMPCMEK sequence also showed similarity to two 4-(cytidine 5'-diphospho)-2-C-

methyl-D-erythritol kinase proteins of Ginkgo biloba (AAZ80385.1 and AAZ80384.1),

corresponding to 70% and 69% identity, respectively.

VMPCMEK and all of the closely related proteins were predicted to have no signal

peptide. Prediction of motifs by Expasy tool revealed three conserved N-myristoylation

sites (GAxxCT, GQxxAV and GSxxTI) shared by VMPCMEK and other closely related

proteins (Figure 24). The software also predicted the presence of another conserved site

in VMPCMEK and the closely related protein sequences which is cAMP- and cGMP-

dependent protein kinase phosphorylation site (RKxT). Besides that, a conserved motif

for ATP-binding site for the functional activity of 4-(cytidine 5'-diphospho)-2-C-methyl-

D- erythritol kinase (Kim et al., 2008) was also detected in the sequence of VMPCMEK

and its closely related proteins (Figure 24).

A phylogenetic tree (Figure 25) was constructed using MEGA software version 4.0

(Tamura et al., 2007) to estimate the genetic relatedness between amino acid sequence of

VMPCMEK with its closely related proteins. From the phylogenetic tree, VMPCMEK

amino acid sequence is not clustered together with 4-(cytidine 5'-diphospho)-2-C-

methyl-D- erythritol kinase proteins from Solanum lycopersicum, Nicotiana

benthamiana, and Cantharanthus roseus.

Page 112: Vanda Mimi Palmer Thesis

112

Figure 24: Alignment of VMPCMEK with Other Closely Related Protein Sequences

Downloaded from the GeneBank Database. The VMPCMEK is aligned with

HbCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Hevea

brasiliensis (BAF98293.1), SlCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol

kinase) of Solanum lycopersicum (AAF87717.1), CrCMEK (4-(cytidine 5'-diphospho)-2-

C-methyl-D-erythritol kinase) of Catharanthus roseus (ABI35992.1), ZmCMEK (4-

(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Zea mays (ACG34338.1) and

NbCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Nicotiana

benthamiana (ABO87658.1). Residues 189 through 204 (highlited in box) are the

conserved ATP-binding site for the functional activity of 4-(cytidine 5'-diphospho)-2-C-

methyl-D-erythritol kinase (Kim et al., 2008). There are three putative conserved motifs

for N-myristoylation site shared by VMPCMEK with other closely related plant proteins

(GAxxCT, GQxxAV and GSxxTI). The motifs are marked with four stars (****).

Besides that, there is another conserved motifs for cAMP- and cGMP-dependent protein

kinase phosphorylation site (RKxT) marked with two stars (**).

RKxT

**

GAxxCT ****

GQxxAV GSxxTI

**** ****

Page 113: Vanda Mimi Palmer Thesis

113

VMPCMEK (Vanda Mimi Palmer)

HbCMEK (Hevea brasiliensis)

SrCMEK (Stevia rebaudiana)

SlCMEK (Solanum lycopersicum)

NbCMEK (Nicotiana benthamiana)

CrCMEK (Catharanthus roseus)

AtCMEK (Arabidopsis thaliana)

ZmCMEK (Zea mays)

GbCMEK1 (Ginkgo biloba 1)

GbCMEK2 (Ginkgo biloba 2)

Figure 25: A Phylogenetic Tree of VMPCMEK with Homologous Proteins. The

phylogenetic tree is constructed by MEGA software version 4.0 using amino acid

sequence of VMPCMEK with the homologous proteins available in GeneBank database

(NCBI) including other 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from

Hevea brasiliensis (BAF98293.1), Stevia rebaudiana (ABB88838.3), Solanum

lycopersicum (AAF87717.1), Nicotiana benthamiana (ABO87658.1), Catharanthus

roseus (ABI35992.1), Arabidopsis thaliana (AAG01340.1) Zea mays (ACG34338.1) and

two proteins from Ginkgo biloba (AAZ80384.1) and (AAZ80385.1).

Page 114: Vanda Mimi Palmer Thesis

114

Relative expression analysis of VMPCMEK in different tissues (Figure 26) of Vanda

Mimi Palmer showed up-regulated expressions in floral tissues in contrast to vegetative

tissues. Among the floral tissues, petal and sepal showed the highest expression of

VMPCMEK transcript which was approximately two folds higher than the bud

(calibrator). In the lip, the expression of VMPCMEK was detected lower than the bud.

Relative expression analysis of VMPCMEK at different floral developmental stages

showed that VMPCMEK was differentially expressed (Figure 27). The expression level

of VMPCMEK increased gradually from young bud to half-open flower (the highest

level), followed by a decrease in fully-open flower stage. Relative expression study of

VMPCMEK at different time points in a 24-hour cycle (12 hours in light and followed by

12 hours in dark) (Figure 28) showed a differential expression pattern. From the analysis,

expression of VMPCMEK increased gradually from 12.00am to 8.00am, followed by a

gradual decrease until 2.00pm (the lowest peak). After 2.00pm, the expression increased

again gradually until 10.00pm.

4.2.2.3 Sequence and Expression Analyses of Putative Cytochrome P450 Protein

(VMPCyP450)

VMPCyP450 has a full-length cDNA transcript of 1785bp comprising 1614bp of ORF,

18bp of 5‟-UTR and 153bp of 3‟-UTR with a poly-A tail (Figure 29). The VMPCyP450

sequence encodes for 538 amino acid residues (Figure 30) with a predicted molecular

weight of 62.1 kD and an isoelectric point (pI) of 8. A homologous sequence

Page 115: Vanda Mimi Palmer Thesis

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0.00

0.50

1.00

1.50

2.00

2.50

bud fully-

open

flower

petal sepal lip leaf root shoot stalk

Tissues

Rela

tive

Exp

ressio

n o

f V

MP

CM

EK

(fo

ld c

han

ge)

Figure 26: Relative Expression Study of VMPCMEK Transcript in Different

Tissues Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open

flower, petal, sepal and lip meanwhile the vegetative tissues used in the expression study

are leaf, root, shoot and stalk. The quantitative expression level of VMPCMEK in each

tissue was calculated relative to the calibrator which was the bud collected at 12.00 noon.

Standard error between three replicates of relative gene expression in each tissue is

indicated by error bar.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

young bud

(green)

mature bud

(red)

half-open

flower

fully-open

flower

14-day old

flower

Developmental Stages

Rel

ativ

e

Exp

ress

ion

of

VM

PC

ME

K

(fo

ld c

han

ge)

Figure 27: Relative Expression Analysis of VMPCMEK Transcript at Different

Developmental Stages During Flower Development. This study was carried out on

five different flower developmental stages of Vanda Mimi Palmer including young bud,

mature bud, half-open flower, fully-open flower and 14-day old fully-open flower.

Quantitative measurement of VMPCMEK expression in each flower developmental stage

was expressed relative to the calibrator which was the young bud collected at 12.00 noon.

Standard error between three replicates of relative gene expression at each developmental

stage is indicated by error bar.

Page 116: Vanda Mimi Palmer Thesis

116

0.00

0.50

1.00

1.50

2.00

2.50

3.00

12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Time

Rela

tive e

xp

ressio

n o

f V

MP

CM

EK

(fo

ld e

xp

ressio

n)

Figure 28: Relative Expression Analysis of VMPCMEK at Different Time Points in

a 24-hour Cycle. Quantitative expression of VMPCMEK at each time point was

expressed relative to the calibrator which was the fully-open flower collected at 12.00

noon. Standard error between three replicates of relative gene expression at each time

point is indicated by error bar.

Page 117: Vanda Mimi Palmer Thesis

117

1 ATA CTG CTG CTG CCA CTA ATG TCT TCT TCC TCA AGC TCC TCA CTT 45

M S S S S S S S L 9

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

VMPCyP450 ORF Forward Primer

46 CTG CCA TAC AAA CTC ATT GCT TTC AGC GCA ATC TTC CTC ATC AGT 90

10 L P Y K L I A F S A I F L I S 24

91 TGG ATC TTC CTT CAT AGA TGG GCA CAG AGA AAC CGC AGA GGT CCG 135

25 W I F L H R W A Q R N R R G P 39

136 AAG ACA TGG CCG CTC ATC GGA GCC GCC ATT GAA CTG CTC AAC AAC 180

40 K T W P L I G A A I E L L N N 54

181 TAT GAA CGC ATG CAT GAT TGG ATT ACA GAT TAC TTG TCT GAA TGG 225

55 Y E R M H D W I T D Y L S E W 69

226 AGG ACT GTT ACT GTC CCC TTG CCC TTC ACT TCA TAC ACT TAT ACT 270

70 R T V T V P L P F T S Y T Y T 84

271 GCA GAC CCT GCA AAT GTG GAG CAT ATT CTG AAG ACC AAC TTC AAC 315

85 A D P A N V E H I L K T N F N 99

316 AAC TAT CCC AAG GGA GAG CTA TTT AGA TCA TAT ATG GAG GTA TTG 360

100 N Y P K G E L F R S Y M E V L 114

361 CTG GGA GAT GGG ATA TTT AAC GCA GAT GGA GAG CTG TGG AGG AAG 405

115 L G D G I F N A D G E L W R K 129

406 CAG AGG AAG ACT GCA AGC TTT GAG TTT GCT TCA AAG AAC TTG AGG 450

130 Q R K T A S F E F A S K N L R 144

451 GAA CTG AGC ACC GTT GTG TTT AGA GAG TAT GCT TTG AAA CTA TCT 495

145 E L S T V V F R E Y A L K L S 159

496 GAC ATA TTA TGC CAA GCC TCT TGC AAA GAT CAT CAT CAA GCT GTA 540

160 D I L C Q A S C K D H H Q A V 174

541 GAT ATT CAG GAT TTA TTC ATG AGG ATG ACA ATG GAC TCC ATA TGC 585

175 D I Q D L F M R M T M D S I C 189

586 AAG CTT GGT TTT GGA GTG GAG ATA GGG ACA CTA TCT CCC CAA CTC 630

190 K L G F G V E I G T L S P Q L 204

631 CCT GAT AAC AGC TTT GCT CGA GCT TTC GAC ACC GCG AAC GCG ACC 675

205 P D N S F A R A F D T A N A T 219

676 GTC ACG CGT CGA TTC TTC GAT CCC TTG TGG AGG TTG AAG AGG TTT 720

220 V T R R F F D P L W R L K R F 234

721 CTT TGT GTG GGA TCA GAG GCT GCC CTC AAC CAA AAT ATC AGA ATT 765

235 L C V G S E A A L N Q N I R I 249

766 GTT AAT GAC TTC ACC TCT AAT GTT ATA CGT ACA AGA AAG GCT GAG 810

250 V N D F T S N V I R T R K A E 264

811 ATC ATG AGA GCT AAA CAA AAC GGG CAT CAT GAT GAG ACA AAG CAA 855

265 I M R A K Q N G H H D E T K Q 279

856 GAC ATA CTA TCA AGG TTC ATC GAG CTC GCC AAC ACC GAC AAA GAG 900

280 D I L S R F I E L A N T D K E 294

901 AGT GAT TTC AGC ACG GAA AAA GGT TTA AGA GAT GTG GTG CTA AAC 945

295 S D F S T E K G L R D V V L N 309

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118

946 TTT GTT ATT GCA GGG AGG GAC ACT ACT GCT GCA ACG CTC TCA TGG 990

310 F V I A G R D T T A A T L S W 324

>>>>>>>>>>>>>>>>>>

VMPCyP450 RT-PCR Forward Primer

991 TTT ATA TAC ATA TTA GTC ACA CAA CCT CAG GTG GCA CAG AAA CTC 1035

325 F I Y I L V T Q P Q V A Q K L 339

>>>>>>

1036 TAT ATA GAG ATG AAA GAG TTT GAG GAG ATC AGA GCT GAA GAA GAA 1080

340 Y I E M K E F E E I R A E E E 354

1081 AAT ATA AAT TTG GAT TTA TGT AAT TTG GAA GAT ATG GAT TCA TTC 1125

355 N I N L D L C N L E D M D S F 369

1126 AGA AAC AGA TTA TCA GAT TTT TCG AGG CTT TTG GAT TAT GAT TCA 1170

370 R N R L S D F S R L L D Y D S 384

<<<<<<<<<<<<<<<<<<<<<<<<<<

VMPCyP450 RT-PCR Reverse Primer

1171 TTA GCA AGG CTG CAA TAT CTG CAT GCA TGC ATT ACA GAG ACC CTG 1215

385 L A R L Q Y L H A C I T E T L 399

1216 AGG CTG TTT CCT CCT GTT CCT CAG GAC GCG AAA GGC ATT TTG AAG 1260

400 R L F P P V P Q D A K G I L K 414

1261 GAT GAT GTT CTC CCT GAC GGA ACA AAA CTG AGA GCC GGG GAA ATG 1305

415 D D V L P D G T K L R A G E M 429

1306 GTG CTA TAC GTC CCC TAT TCA ATG GGA AGA ATG GAG TAC ATT TGG 1350

430 V L Y V P Y S M G R M E Y I W 444

1351 GGC ATC GAC GCA TCA GAA TTT CGC CCC GAA AGA TGG CTA AAT AAC 1395

445 G I D A S E F R P E R W L N N 459

1396 GAC AAT AAT TCC GTC CAA AAT AAC GTC TCT CCA TTC AAG TTC ACG 1440

460 D N N S V Q N N V S P F K F T 474

1441 GCG TTT CAG GCT GGT CCC AGA ATG TGC TTG GGG AAG GAC TCC GCT 1485

475 A F Q A G P R M C L G K D S A 489

1486 TAT CTG CAG ATG AAG ATG ACA GCA GCG TTA CTC TGC AGG TTC TTT 1530

490 Y L Q M K M T A A L L C R F F 504

1531 CAA TTC AGA CTT GCT CCT CAT CAT CCT CCT GTT AAG TAT AGG ATG 1575

505 Q F R L A P H H P P V K Y R M 519

1576 ATG ATA GTA CTT TCC ATG GCG CAT GGC CTG CAT GTG CTC GTT TGT 1620

520 M I V L S M A H G L H V L V C 534

1621 AGA AGA GGA TCA TGA TTT TTG ATG CAT GGA TCT ATG TTT TAT TAT 1665

535 R R G S * 538

1666 TAA GTT ATT GCG TCT GTT TGT TGT CCA TAA GTG AAG TGC AGA CAA 1710

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

VMPCyP450 ORF Reverse Primer

1711 CAT CAT TAT TAT ATG TCA TGC CCC CTA AAT GTT TAC TTC CGA AAA 1755

1756 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1785

Figure 29: The Nucleotide and Deduced Amino Acid Sequence of VMPCyP450

(Putative Cytochrome P450 Protein). The open reading frame (ORF) of VMPCyP450

starts at nucleotide 19. The asterisk (*) indicates stop codon. The putative

polyadenylation signal is bold and underline. The location of gene specific primers used

in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).

Page 119: Vanda Mimi Palmer Thesis

119

Figure 30: PCR Product of Open Reading Frame (ORF) of VMPCyP450 of Vanda

Mimi Palmer. The ORF represents 538 amino acids. The PCR product was

electrophoresed on 1% (w/v) agarose gel. Lane M: 100bp marker (Vivantis, Malaysia);

Lane 1: ORF of clone VMPCyP450.

1500bp

500bp

M 1

~1600bp

Page 120: Vanda Mimi Palmer Thesis

120

analysis by BLASTX and BLASTP (NCBI) shows that VMPCyP450 exhibited similarity

to sequences that encode cytochrome P450 proteins from other plants (39-67% identity).

From the BLASTX and BLASTP analyses, VMPCyP450 has the closest similarity to

putative cytochrome P450 protein of Oryza sativa (ABF 94184.1), and cytochrome P450

protein of Populus trichocarpa (EEE89622.1), corresponding to 66% identity. Besides

that, VMPCyP450 also showed similarity to cytochrome P450 monooxygenase of

Petunia hybrida (AAZ39646.1) (45% identity) and cytochrome P450 monooxygenase

from other plants.

Localizome analysis predicts that VMPCyP450 protein does not have any signal peptide.

Most of other plant cytochrome P450 proteins were predicted by the Localizome software

to have signal peptide except cytochrome P450 protein of Arabidopsis thaliana,

cytochrome P450 protein of Populus trichocarpa and cytochrome P450 monooxygenase

of Petunia hybrida. VMPCyP450 and all the closely related proteins were predicted to

have a transmembrane domain by the Localizome software. The deduced VMPCyP450

protein was predicted to have 11 amino acids at the N-terminal (non-cytosolic), 21 amino

acids located in the membrane and 506 amino acids in cytosol. Expasy programme

prediction shows that three N -myristoylation sites (GxxxAD, GVxxGT and GsxxAL)

were shared by VMPCyP450 and its closely related proteins (Figure 31). Besides that,

there was another conserved site for tyrosine kinase phosphorylation (see Figure 31). A

phylogenetic tree (Figure 32) was constructed using the MEGA version 4.0 (Tamura et

al., 2007) to estimate the genetic relationship of VMPCyP450 with other cytochrome

Page 121: Vanda Mimi Palmer Thesis

121

Figure 31: Alignment of VMPCyP450 with Other Closely Related Protein Sequences

Downloaded from GeneBank Database NCBI. The VMPCyP450 was aligned with

cytochrome P450 of Ricinus communis (EEF39957.1) putative cytochrome P450 of

Arabidopsis thaliana (AAG60111.1), putative cytochrome P450 protein of Oryza sativa

from Japonica Cultivar (AAL84318.1), cytochrome P450 of Zea mays (ACG35470.1)

and cytochrome P450 monooxygenase of Petunia hybrida (AAZ39646.1). There are

three N-myristoylation sites (GxxxAD, GVxxGT and GsxxAL) shared by VMPCyP450

with other closely related proteins which are marked with four stars (****). There is

another putative conserved region for tyrosine kinase phosphorylation site (RxxExxxY)

marked with five delta symbol (∆∆∆∆∆

).

GIxxAD

****

GSxxAL

****

RxxExxxY ∆∆∆∆∆

GVxxGT

****

Page 122: Vanda Mimi Palmer Thesis

122

VMPCyP450 (Vanda Mimi Palmer)

PhP450 (Petunia x hybrida)

MtP450 (Medicago truncatula)

GmP450 (Glycine max)

RcP450 (Ricinus communis)

PtP450 (Populus trichocarpa)

AtP450 (Arabidopsis thaliana)

CaP450 (Capsicum annuum)

OsP450 (Oryza sativa-japonica cultiva...

ZmP450 (Zea mays)

Figure 32: A Phylogenetic Tree of VMPCyP450 and Homologous Proteins. The

phylogenetic tree is constructed by MEGA software version 4.0 using amino acid

sequence of VMPCyP450 with the homologous proteins available in GeneBank database

(NCBI) including cytochrome P450 of Ricinus communis (EEF39957.1), cytochrome

P450 of Populus trichocarpa (EEE89622.1) cytochrome P450 of Arabidopsis thaliana

(AAG60111.1), cytochrome P450 of Capsicum annuum (ACD10924.1), cytochrome

P450 protein of Oryza sativa from Japonica Cultivar (AAL84318.1), cytochrome P450 of

Zea mays (ACG35470.1), cytochrome P450 monooxygenase of Petunia hybrida

(AAZ39646.1), cytochrome P450 monooxygenase of Medicago tranculata

(ABC59094.1) and cytochrome P450 monooxygenase of Glycine max (Soy

bean)(ABC68403.1).

Page 123: Vanda Mimi Palmer Thesis

123

P450 proteins. The phylogenetic tree shows that VMPCyP450 amino acid sequence was

clustered together with cytochrome P450 monooxygenase of Medicago truncatula,

Glycine max and Petunia hybrida and supported by the bootstrap value of 99 for the

branch.

Relative expression analysis in different tissues (Figure 33) of Vanda Mimi Palmer

including floral and vegetative tissues by real-time RT-PCR shows that the VMPCyP450

had up-regulated expression in floral tissues especially in the lip. The other floral tissues

including petal and sepal showed lower expressions of VMPCyP450 compared to the lip.

For vegetative tissues such as leaf, root, shoot, and stalk, the expression of VMPCyP450

was detected at a very low level. Expression analysis of VMPCyP450 transcript at

different flower developmental stages (Figure 34) shows a developmentally regulated

pattern. Meanwhile, the expression analysis of VMPCyP450 at different time points in a

24-hour cycle (Figure 35) also shows a differential expression pattern.

4.2.2.4 Sequence and Expression Analyses of Unknown protein (VMPA28)

The full-length sequence of VMPA28 cDNA transcript is 972bp containing 591bp ORF

flanked with 148bp 5‟UTR and 233bp 3‟UTR including a poly-A tail (Figure 36). The

ORF encodes a protein of 197 amino acid residues (Figure 37). Expasy tool (Prolite) has

predicted 22.32kD as the molecular mass of VMPA28 protein with an isoelectric point of

9.06. The Expasy tool also predicts the presence of a N-glycosylation site (NTSN), two

Page 124: Vanda Mimi Palmer Thesis

124

0.00

1.00

2.00

3.00

4.00

5.00

6.00

bud fully-

open

flower

petal sepal lip leaf root shoot stalk

Tissues

Rel

ativ

e

Exp

ress

ion

of

VM

PC

yP45

0

(fo

ld c

han

ge)

Figure 33: Relative Expression Analysis of VMPCyP450 in Different Tissues

Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open flower,

petal, sepal and lip meanwhile the vegetative tissues used in the expression study are leaf,

root, shoot and stalk. The quantitative expression level of VMPCyP450 in each tissue

was calculated relative to the calibrator which was the bud collected at 12.00 noon.

Standard error between three replicates of relative gene expression in each tissue is

indicated by error bar.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

young bud

(green)

mature bud

(red)

half-open

flower

fully-open

flower

14-day old

flower

Developmental Stages

Rel

ativ

e

Exp

ress

ion

of

VM

PC

yP45

0

(fo

ld c

han

ge)

Figure 34: Relative Expression Study of VMPCyP450 at Different Flowering

Developmental Stages. This study was carried out on five flower developmental stages

of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open

flower and 14-day old fully-open flower. Quantitative measurement of VMPCyP450

expression in each flower developmental stage was expressed relative to the calibrator

which was the young bud collected at 12.00 noon. Standard error between three replicates

of relative gene expression at each developmental stage is indicated by error bar.

Page 125: Vanda Mimi Palmer Thesis

125

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Time

Rela

tive E

xp

ressio

n o

f V

MP

CyP

450

(fo

ld e

xp

ressio

n)

Figure 35: Relative Expression Study of VMPCyP450 at Different Time Points in a

24-hour Cycle. Quantitative expression of VMPCyP450 at each time point was

expressed relative to the calibrator which was the fully-open flower collected at 12.00

noon. Standard error between three replicates of relative gene expression at each time

point is indicated by error bar.

Page 126: Vanda Mimi Palmer Thesis

126

2 AAA AAC GTC GTT GTG TCT CGG GGT CGT TGG GGA GAA TTT CTT AAT 46

47 AAC AGT CGG AAA AAA GGT TCC CTA ATG ATA AGC GGG ACA GTT AGC 91

92 GCA ACT AAA TTA ATG TGA GAT TAG TTC AAT TCT TAG GCA CCC CAG 136

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

VMPA28 ORF forward Primer

137 ATT TTA CAT TTT ATG TTT TCC GGT TCG TTT TTT TTG GGA AAT TTT 181

M F S G S F F L G N F 11

182 TTA GTG AAT AAC AAT TTC ACC CAG GAA AAC AGT TAT GAC CAT GAT 226

12 L V N N N F T Q E N S Y D H D 26

227 TAC GCC AAG CTT GCA TGC CTG CAG GTT GAC TTT AGA GGG GAT CCA 271

27 Y A K L A C L Q V D F R G D P 41

272 AAT TTT AAA TAT CCC GCA ACC TGT CCC ACC TTT TCT CCC CTT TTC 316

42 N F K Y P A T C P T F S P L F 56

317 GTC TCT TTT TCC CTG TTT CTC TTT CTC ATC CGA TTG ATG GAT CTT 361

57 V S F S L F L F L I R L M D L 71

362 AGG GCG GCG ATA GTC GCC GCC GCC GGC GAG CGT TGG ACG GAG GAG 406

72 R A A I V A A A G E R W T E E 86

407 CGG CAC TCC CGC TTC CTC AAC TCG ATC GAA AGT ACT TTC GTC CAT 451

87 R H S R F L N S I E S T F V H 101

452 CAA ATG CTC GGC ATC CAT CCC GAC GGC GAT AAC CTC CGC CGA TGC 496

102 Q M L G I H P D G D N L R R C 116

497 GCG GCG AGG CTC GAC CGT CGT GTT CCC GAT TGC ATC GCC GGT AAA 541

117 A A R L D R R V P D C I A G K 131

542 GAG TCT GCG AAG AGT TCT CAG ATG CGA TCG CCG GAT AGG AGG CCT 586

132 E S A K S S Q M R S P D R R P 146

587 GCT GCC ATT ACT GCG GGC GCC AAC ACC TCT AAT TGT ACA CGG AAA 631

147 A A I T A G A N T S N C T R K 161

>>>>>>>>>>>>>>

632 CGA TCA CTG CGG CGA TAT GAT GCG TCG CTA GAC CAG GTG GTG CCG 676

162 R S L R R Y D A S L D Q V V P 176

>>>>>>>>>>>

VMPA28 RT-PCR Forward Primer

677 GAG TTC AAA AAT AAG AAC GTC GGC GAG GAT GCA TCC AAG CGA AAG 721

177 E F K N K N V G E D A S K R K 191

722 TTT GAA GAC GCA GCA CAT TAA CAA TGA GAA CAA TAT TGG AGT CTA 766

192 F E D A A H * 197

767 AAG CTG CAG CTG TCT TCA TCT CTT GCT GTC TAC AAG CAA TGT TTG 811

<<<<<<<<<<<<<<<<<<<<<<<<<<< VMPA28 ORF Reverse Primer

812 ACT TGC ATG TTG AAA GGA ATG TAG AAA TGT GTT CTT ATA TAT TAT 856

<<<<<<<<<<<<<< VMPA28 RT-PCR Reverse Primer

<<<<<<<<<<<<<<

857 GTT TTA GAG GAA ACG TAT TTA TGA TAA TTT TTG CTT GTT AAA GTT 901

902 GGT TTT TGC CAT TTC AAC ATG GTT TCA TTC TGT GAA CAT TTT AGA 946

947 TCA AAA AAA AAA AAA AAA AAA AAA AAA 973

Figure 36: The Nucleotide and Deduced Amino Acid Sequences of VMPA28. The

open reading frame (ORF) of VMPA28 starts at nucleotide 149. The asterisk (*) indicates

stop codon. The putative adenylation signal is bold and underline in the nucleotide

sequence. The location of gene specific primers used in real-time PCR analysis and ORF

isolation are shown by arrow heads (>>>> and <<<<).

Page 127: Vanda Mimi Palmer Thesis

127

Figure 37: PCR Product of Open Reading Frame (ORF) of an Unknown Protein of

Vanda Mimi Palmer (VMPA28). The PCR product was electrophoresed on 1% (w/v)

agarose gel. Lane M: 100bp marker (Vivantis); Lane 2: ORF of clone VMPA28.

1000bp

500bp

M 1 2

~ 700bp

Page 128: Vanda Mimi Palmer Thesis

128

protein kinase C phosphorylation sites (SxK, SxR) and a N-myristoylation site

(GAxxSN). BLASTX and BLASTP analyses (NCBI) show the deduced amino acid of

VMPPA28 had no significant similarity to any known sequence in the GeneBank

database. Localizome analysis shows there was no signal peptide in the deduced protein

sequence of VMPA28.

Expression analysis of VMPA28 was carried out in different tissues, at different flower

developmental stages and also at different time points in a 24-hour cycle by real-time RT-

PCR. Analysis of VMPA28 transcript in different tissues (Figure 38) shows a slight up-

regulated expression in floral tissues compared to vegetative tissues. For analysis of

VMPA28 transcript at different flower developmental stages (Figure 39), the expression

of VMPA28 was developmentally-regulated where the transcripts level increased

gradually from the bud to the fully-open flower stage, followed by a gradual decrease

until the end of flower life-time. The transcript was also found to be differentially

expressed at different time points in a 24-hour cycle (Figure 40).

Page 129: Vanda Mimi Palmer Thesis

129

0.00

0.40

0.80

1.20

1.60

bud fully-

open

flower

petal sepal lip leaf root shoot stalk

Tissues

Rela

tive

Exp

ressio

n o

f V

MP

A28

(fo

ld c

han

ge)

Figure 38: Relative Expression Study of VMPA28 Transcript in Different Tissues

Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open flower,

petal, sepal and lip meanwhile the vegetative tissues used in the expression study are leaf,

root, shoot and stalk. The quantitative expression level of VMPA28 in each tissue was

calculated relative to the calibrator which was the bud collected at 12.00 noon. Standard

error between three replicates of relative gene expression in each tissue is indicated by

error bar.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

young bud

(green)

mature bud

(red)

half-open

flower

fully-open

flower

14-day old

flower

Developmental Stages

Rel

ativ

e E

xpre

ssio

n o

f V

MP

A28

(fo

ld c

han

ge)

Figure 39: Relative Expression Study of VMPA28 at Different Flower

Developmental Stages. The study was carried out in five flower developmental stages

of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open

flower and 14-day old fully-open flower. Quantitative measurement of VMPA28

expression in each flower developmental stage was expressed relative to the calibrator

which was the young bud collected at 12.00 noon. Standard error between three replicates

of relative gene expression at each developmental stage is indicated by error bar.

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130

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Time

Rela

tive E

xp

ressio

n o

f V

MP

A28

(fo

ld c

han

ge)

Figure 40: Relative Expression Study of VMPA28 at Different Time Points in a 24-

hour Cycle. Quantitative expression of VMPPAAS at each time point was expressed

relative to the calibrator which was the fully-open flower collected at 12.00 noon.

Standard error between three replicates of relative gene expression at each time point is

indicated by error bar.

Page 131: Vanda Mimi Palmer Thesis

131

CHAPTER 5

DISCUSSIONS

5.1 Biochemical Analysis of the Scent of Vanda Mimi Palmer

Biochemical analysis of the scent of Vanda Mimi Palmer by gas chromatography-mass

spectrometry (GC-MS) showed a fluctuating pattern of volatile emission by fully-open

flower of Vanda Mimi Palmer in a 24-hour cycle with the highest level detected during

the daytime (in light) but none at night (in dark). This emission pattern was observed in

most roses and snapdragon (Anthirrhinum majus) whereby the highest level of scent

emission was reported to occur during daytime (Helsper et al., 1998; Picone et al., 2004;

Dudareva et al., 2003). Meanwhile, for some other plants such as in Petunia hybrida and

Stephanotis floribunda, the highest level of scent emission occurs at night (Verdonk et

al., 2003; Pott et al., 2002). The pattern of scent emission of Vanda Mimi Palmer in a 24-

hour cycle might be controlled by some factors such as photoperiod and circardian clock

like in other scented plants (Lu et. al, 2002). Besides that, flowers of Vanda Mimi Palmer

also showed a developmentally regulated pattern of scent emission whereby no volatile

compound was detected in the bud stage. Emission of volatile compounds was detected

from the half-open flower stage and increased to the maximum level in the fully-open

flower stage. The same pattern of scent emission was previously reported in other scented

flowers including Clarkia breweri and Anthirrhinum majus (Pichersky et al., 1994;

Nagegowda et al., 2008).

Page 132: Vanda Mimi Palmer Thesis

132

Based on the GC-MS analysis of the scent of Vanda Mimi Palmer, volatile compounds

emitted by the flowers are derived from the terpenoid, benzenoid and phenylpropanoid

groups. Thus, there are possibly two main pathways involved in the fragrance

biosynthesis of Vanda Mimi Palmer which are terpenoid as well as benzenoid and

phenylpropanoid pathways. Hsiao et al. (2006) reported that the volatile compounds in

Phalaenopsis bellina (a scented orchid from Taiwan) are derived from the three

pathways: terpenoid, lipoxygenase, as well as benzenoid and phenylpropanoid pathways

(Hsiao et al., 2006). These three fragrance biosynthetic pathways were also reported in

other well studied scented flowers like Rosa hybrida (Guterman et al., 2002), Clarkia

breweri (Pichersky et al., 1995), Petunia hybrida (Boatright et al., 2004) and

Anthirrhinum majus (Nagegowda et al., 2008).

The GC-MS analysis of the scent of Vanda Mimi Palmer showed that there are four

volatile compounds (linalool, ocimene, linalool oxide and nerolidol) which might have

been derived from the terpenoid pathway. Linalool, ocimene and linalool oxide are

classified as monoterpenes (Croteau and Karp, 1991; Knudsen and Gershenzon, 2006)

while nerolidol is classified as a sesquiterpene (Knudsen and Gershenzon, 2006;

Nagegowda et al., 2008). Monoterpenes and sesquiterpenes are the common compounds

in the scent of scented orchids including Phalaenopsis bellina (Hsiao et al., 2006),

Dendrobium beckleri and Phalaenopsis violacea (Kaiser, 1993). The terpenoid

compounds emitted by Vanda Mimi Palmer‟s flower were also detected in the scent of

other scented flowers including Anthirrhinum majus (Linalool, ocimene and nerolidol)

(Dudareva et al., 2003; Nagegowda et al., 2008), and Clarkia breweri (Linalool)

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(Pichersky et al., 1994). Thus, the terpenoid pathway in Vanda Mimi Palmer involved in

the biosynthesis of linalool, ocimene and nerolidol compounds might be closely related to

the terpenoid pathway in other well studied scented flowers. The terpenoid pathway for

the floral scent biosynthesis in those well studied scented flowers could be used for the

study of the terpenoid pathway in Vanda Mimi Palmer.

Besides terpenoids, there were four other compounds in the scent of Vanda Mimi Palmer

which might be derived from the benzenoid and phenylpropanoid pathway such as

methylbenzoate, benzyl acetate, phenylethanol and phenylethyl acetate. The same

benzenoid and phenylpropanoid compounds were reported to be present in the scent of

other scented orchids like Dendrobium trigonopus, Dendrochilum cobbianum and Vanda

tessellata (Kaiser, 1993), and in other scented flowers like Rosa hybrida (Shalit et al.,

2003), Petunia hybrida (Verdonk et al., 2003), Clarkia breweri (Raguso and Pichersky,

1995; Dudareva et al., 1998) and Anthirhinum majus (Dudareva et al., 2000). In

Phalaenopsis bellina, some other benzenoid and phenylpropanoid compounds such as 3-

phenyl-2-propen-1-ol, 3-methylphenyl butanoic ester, and 2-methylphenyl butanoic ester

were detected in its floral scent (Hsiao et al., 2006). Benzyl acetate which was detected in

the scent of Vanda Mimi Palmer, was also reported to be present in the scent of Rosa

hybrida (Shalit et al., 2003) and Clarkia breweri (Raguso and Pichersky, 1995; Dudareva

et al., 1998). Another detected compound, methylbenzoate was also reported in many

scented flowers like Petunia hybrida (Verdonk et al., 2003) and Anthirhinum majus

(Dudareva et al., 2000). Besides that, phenylethanol, a compound which is also present in

the scent of Vanda Mimi Palmer, was identified in the scent of Rosa hybrida (Shalit et

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al., 2003), Petunia hybrida (Verdonk et al., 2003), Clarkia breweri (Raguso and

Pichersky, 1995; Dudareva et al., 1998), and Rosa hybrida (Shalit et al., 2003). Thus, the

benzenoid and phenylpropanoid pathways in Vanda Mimi Palmer which could possibly

be involved in the biosynthesis of methylbenzoate, benzyl acetate, phenylethyl acetate

and phenylethanol compounds might be closely related to the benzenoid and

phenylpropanoid pathways in well studied scented flowers especially like Petunia

hybrida, Clarkia breweri and Rosa hybrida.

Solid phase Micro-extraction (SPME) method used in this study is one of the advanced

methods utilised to capture volatile compounds emitted by scented flowers. In this study,

a modified funnel was used to collect and accumulate the volatiles emitted by Vanda

Mimi Palmer flowers in a special trap before being captured by the SPME. It is possible

that there might be some other compounds in the scent of Vanda Mimi Palmer emitted in

traces amount that could not be detected in this study using the above method (capturing

was just for 15 minutes). Trace compounds could be detected and identified using a

combination of modern headspace attached directly to the GC-MS where the volatiles

emitted by the scented flowers are accumulated in a special chamber for 2-3 hours and

then concentrated with a special pump prior injection into the GC-MS port.

Unfortunately, this approach was not employed in this study due to inavailability of the

system in Universiti Putra Malaysia. There is another method used in the floral scent

studies in rose by Hendel-Rahmanim et al. (2007) and Farhi et al. (2010) on Rosa hybrida

whereby a few grams of the rose petals were soaked in hexane for a few hours. The

debris was removed and the supernatant containing hexane with the extracted compounds

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was filtered and then concentrated with nitrogen before being injected into the GC-MS

injector port. In this study, the above mentioned method was applied to extract the

essential oil from Vanda Mimi Palmer using hexane and a large amount of flowers

(100grams). Most of the compounds detected at high levels in the scent of Vanda Mimi

Palmer were also detected in the essential oil of Vanda Mimi Palmer. However, there

were some compounds including ocimene and nerolidol which were not found in the

essential oil. One possible reason for non-detectability of ocimene is because the

compounds might not be stored in the cell long enough prior to its release into the air. As

for nerolidol, this compound could be synthesized at very low amount compared to other

compounds such as linalool, methylbenzoate, benzyl acetate and phenylethyl acetate in

the floral tissues of Vanda Mimi Palmer. Besides that, a lot of other compounds which

are not emitted by fully-open flower of Vanda Mimi Palmer were also detected in the

essential oil (see Appendix D, subsection (c) and (d)). This could be due to the nature of

hexane itself as a powerful solvent to extract out non-polar compounds from the plant

samples.

In Vanda Mimi Palmer, the emitted scent is dominated by ocimene and linalool

compounds in the morning (8.00am-12.00 noon). At this time, other compounds were

detected at very low levels especially the compounds derived from the benzenoid and

phenylpropanoid pathways. However, the emission levels of benzenoid and

phenylpropanoid compounds especially benzyl acetate and phenylethyl acetate increased

drastically after 12.00 noon, higher than the levels of the terpenoid compounds (linalool

and ocimene). Thus, the percentage of each compound of the scent of Vanda Mimi

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Palmer is different at different time points during daytime. This might be the special

characteristic of the scent of Vanda Mimi Palmer to attract different pollinators at

different time points during daytime. Unfortunately no comparison can be made with

other well studied scented flowers such as Petunia hybrida, Clarkia breweri, Antirrhinum

majus and Rosa hybrida as their percentage of each compound in the scent at different

time points and their functions to attract specific pollinators at different time points were

not investigated.

Comparison of the volatiles emitted by Vanda Mimi Palmer (scented orchid) and its

parents, Vanda Tan Chay Yan (non-scented orchid) and Vanda tessellata (scented orchid)

in Table 6 (section 4.1.1) shows ocimene was the only compound detected in the scent of

Vanda Mimi Palmer and both of its parents. Ocimene is a monoterpene derived from the

terpenoid pathway (Dudareva et al., 2003). An ocimene synthase from Vanda Mimi

Palmer is postulated to be involved in the final step of terpenoid biosynthetic pathway.

This enzyme could be involved in catalyzing the formation of ocimene from geranyl

diphosphate, a precursor for monoterpenoid biosynthesis. The transcript of this enzyme

has yet to be identified in Vanda Mimi Palmer. For another monoterpene compound

which is linalool, a linalool synthase transcript has been identified in the floral ESTs of

Vanda Mimi Palmer (Teh Siow Ling, Master student, Faculty of Biotechnology and

Biomolecular Sciences, Universiti Putra Malaysia, pers. comm. on 18th September 2008).

This enzyme might be involved in catalyzing the formation of linalool from geranyl

diphosphate. The linalool synthase gene might be derived from Vanda tessellata since

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linalool was also detected in the scent of Vanda tessellata previously by Kaiser (Kaiser,

1993) using GC-MS.

Besides that, the formation of nerolidol (a sesquiterpene) was postulated to be catalyzed

by sesquiterpene synthase (Chan, 2009). The transcript of sesquiterpene synthase was

identified from the floral ESTs of Vanda Mimi Palmer. This sesquiterpene gene in Vanda

Mimi Palmer could be contributed by the gene pool from Vanda Tan Chay Yan since

coupane, a sesquiterpene was identified to be emitted by fully-open flower of Vanda Tan

Chay Yan (see Figure 10, in section 4.1.1). In contrast, no sesquiterpene was detected in

the scent of Vanda tessellata (another parent of Vanda Mimi Palmer) as reported by

Kaiser (Kaiser, 1993). The combination of GC and MS used by Kaiser in his work

(Kaiser, 1993) at that time was sensitive enough to detect the presence of sesquiterpenes

since some sequiterpenes were detected in other scented orchids including Aerangis

confuse (germacrane D and nerolidol), Dendrobium virgineum (nerolidol), Laelia anceps

(nerolidol), Oncidium longipes (nerolidol), and Polystachya fallax (caryophyllene and α-

farnesene) (Kaiser, 1993). In Vanda tessellata, sesquiterpene synthase might not be

expressed if it is present as a recessive allele of the gene. While in Vanda Tan Chay Yan,

seisquiterpene synthase gene might be represented by a dominant allele. The hybrid of

these orchids could bring heterozygous dominant characteristic of the sesquiterpene

synthase. Interestingly, there were another two sesquiterpenes identified in the essential

oil of of Vanda Mimi Palmer (germacrene and copaene) (see Table 6) besides nerolidol

that was identified in the scent of Vanda Mimi Palmer.

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Volatiles comparison between Vanda Mimi Palmer and its parents (see Table 4, in

section 4.1.1) shows that two benzenoids (benzyl acetate and methylbenzoate) were

detected in the scent of both Vanda Mimi Palmer and Vanda tessellata (Kaiser, 1993).

These compounds could be derived from the benzenoid pathway. A number of

benzenoids identified in the scent of Vanda tessellata (Kaiser, 1993) were not detected in

the scent of Vanda Mimi Palmer such as benzaldehyde, benzyl alcohol, cinnamyl alcohol,

methyl cinnamate and methyl salicylate. Similarly, some compounds were only detected

in the scent of Vanda Mimi Palmer including phenylethanol and phenylethyl acetate.

Based on the differences, it was postulated that some modifications might have occurred

in the benzenoid and phenylpropanoid pathways of Vanda Mimi Palmer compared to its

parent Vanda tessellata which showed emission of benzenoids and phenylpropanoids

(Kaiser, 1993). Benzaldehyde, benzyl alcohol and cinnamyl alcohol which were detected

in the scent of Vanda tessellata as final products might be intermediates or precursors for

methylbenzoate, benzylacetate and phenylethyl acetate biosynthesis in Vanda Mimi

Palmer. In addition, phenylethanol compound was identified in the essential oil of both

Vanda Mimi Palmer (scented orchid) and Vanda Tan Chay Yan (non-scented orchid).

The phenylethanol might be used directly or acts as an intermediate for other

phenylpropanoids involved fragrance and non-fragrance metabolism. In addition, the

benzenoid and phenylpropanoid pathway has been involved in the biosynthesis of other

non-fragrance compounds including flavanoids and high molecular weight

phenylpropanoids (Lacombe et al., 1997; Shirley, 2001; Boerjan et al., 2003; Takashi et

al., 2007; Derikvand et al., 2008). Interestingly, in Vanda Mimi Palmer‟s essential oil, the

phenylethanol compound was detected at high level, 11.18% of the total essential oil. In

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Vanda Mimi Palmer, the phenylethanol emission was only detected at trace level during

the highest peak, while the other phenylpropanoid which is phenylethyl acetate was

detected to be emitted during day time at very high level (see Figure 7). This suggested

that phenylethanol might be the main precursor for phenylethyl acetate in Vanda Mimi

Palmer.

Most of the compounds in the scent of Vanda Mimi Palmer were also detected in its

essential oil including linalool, methylbenzoate, benzyl acetate, phenylethanol and

phenylethyl acetate. The compounds might be stored in special oil glands or trichomes

for few hours before they are released into the air as volatiles since a lot of oil glands or

trichomes were detected on the surface of the petal and sepal of fully-open flowers of

Vanda Mimi Palmer (Janna Ong Abdullah, unpublished data). Besides that, two

intermediates of the scent‟s compound such as benzyl alcohol and benzyl benzoate were

detected in the essential oil of Vanda Mimi Palmer (see Table 6, in section 4.1.1). The

intermediate compounds might be the precursors for the production of benzyl acetate and

phenylethyl acetate which were detected in the scent of Vanda Mimi Palmer. In addition,

none of the fragrance compounds in the scent of Vanda Mimi Palmer was detected in the

essential oil of Vanda Tan Chay Yan except for phenylethanol. This phenylethanol might

be involved in non-fragrance activities since the compound was not detected in the

volatiles of fully-open flowers of Vanda Tan Chay Yan. In the essential oils of Vanda

Mimi Palmer and Vanda Tan Chay Yan, a lot of non-fragrant compounds were identified

(see Appendix D, subsection (c) and (d)). This might be due to the nature of the hexane

extraction method itself which could extract out most of the non-polar compounds from

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the sample. Alternatively, pure essential oil could be isolated by a technique called

hydro-distillation. However, the hydro-distillation method has its limitation since it

requires a large amount of flowers.

Based on biochemical analysis on volatiles of Vanda Mimi Palmer and comparison with

its parents‟ scent compounds, it seems that the fragrant characteristic of Vanda Mimi

Palmer might be contributed by a pool of genes from both parents. In other scented

flower especially roses, the scent produced by Rosa chinensis (Chinese rose), an ancestor

of modern roses (Rougetel, 1988) is different from the scent of modern rose cultivars

(Wu et al., 2004). A lot of compounds in the scent of the Chinese rose are not found in

modern roses cultivars and some of them do not have any fragrance because breeding

was only focused on the beautiful colour and shape of the flower instead of the fragrance

itself (Yomogida, 1992; Zuker et al., 1998; Wu et al., 2004). For example, 1,3,5-

trimethoxybenzene compound (TMB) synthesized from ploroglucinol, was identified in

the scent of Rosa chinensis but was not detected in the scent most of modern rose

varieties but a related compound which is 3,5-dihyroxytoulene synthesized from orcinol

was detected in many modern roses. Biochemical modifications could have occured in

the fragrance biosynthetic pathway of roses by interaction of genes and enzymes derived

from parents of rose hybrids (Flament et al., 1993; Lavid et al., 2002; Wu et al., 2004).

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5.2 Molecular Studies of Vanda Mimi Palmer

5.2.1 Isolation of Putative Fragrance-related cDNAs

Thirteen putative fragrance-related cDNA clones were isolated from the floral cDNA

library of Vanda Mimi Palmer and preliminarily characterized by reverse-Northern

analysis with two different probes representing mRNAs of fully-open flower and bud

stage separately. The aim was to select cDNAs with up-regulated expression in fully-

open flower stage (high fragrance emission) compared to bud stage (no fragrance

emission) of Vanda Mimi Palmer which might be potential fragrance-related cDNAs.

From the verification of the putative fragrance-related cDNAs with up-regulated

expression in fully-open flower compared to bud of Vanda Mimi Palmer in section

4.2.1.4, VMPCMEK (putative 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase)

was selected for further characterization because it showed higher expression in fully-

open flower of Vanda Mimi Palmer compared to the bud. This could be due to the

involvement of VMPCMEK transcripts in the early steps of the terpenoid pathway (see

Figure 41) for the biosynthesis of monoterpenes and sesquiterpenes such as linalool,

ocimene and nerolidol as reported in section 4.1.1.

Besides VMPCMEK, VMPCyP450 was also selected for further characterization because

of its high expression in the fully-open flower of Vanda Mimi Palmer compared to the

bud. In the bud, this VMPCyP450 might be involved in other metabolism especially for

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PLASTID CYTOSOL

Figure 41: Elucidation of Terpenoid Pathway in Vanda Mimi Palmer. The pathway is

elucidated based on the terpenoid pathway of Anthirrhinum Majus (Nagegowda et al.,

2008). VMPCMEK and VMPCyP450 which was identified and isolated in this study are

shown in circles. Other putative fragrance related cDNAs that were isolated from floral

ESTs of Vanda Mimi Palmer are shown in boxes (Janna Ong Abdullah, Associate

Professor, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra

Malaysia, pers. comm. on 26th October 2009).

Pyruvate + glyceraldehyde 3-phosphate

1-Deoxy-D- 5-phosphate (DXP)

2-C-Methyl-D-erythritol 4-phosphate (MEP)

4-(Cytidine 5‟-diphospho)-2-C-

Methyl-D-erythritol (CDP-ME)

4-(Cytidine 5‟-diphospho)-2-C-

Methyl-D-erythritol 2-phosphate

(CDP-ME)

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate

(cMEPP)

1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate

(HMBPP)

3S-Hydroxy-3-methylglutaryl-CoA

(HMG-CoA)

3R-Mevalonic acid (MVA)

Mevalonic acid 5-phosphate

Mevalonate diphosphate

Dimethylallyl

diphosphate

(DMAPP)

Isopentenyl

diphosphate (IPP)

Dimethylallyl

diphosphate

(DMAPP)

Isopentenyl

diphosphate

(IPP)

Geranyl diphosphate (GPP)

Farnesyl diphosphate (FPP)

Linalool Ocimene

Linalool oxide Nerolidol

VMPCMEK

VMPCyP450

VMPLIS

VMPHMGR

VMPDXS

VMPDXR

VMPHDS

VMPFPPS

VMPSQS

VMPAACT

2 Acetyl-CoA Acetoacetyl-CoA

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the biosynthesis of non-fragrance compounds since cytochrome P450 has been reported

to be involved in metabolism in the lipoxygenase pathway and also phenolic metabolism

(Ehlting et al., 2006). Another transcript selected for further characterization is an

unknown protein designated as VMPA28. The verification result shows the expression of

VMPA28 transcript was slightly higher in the fully-open flower compared to the bud,

suggesting this transcript might be involved in other metabolisms in the plant system

which has yet to be investigated.

5.2.2 Molecular Characterization of Putative Fragrance-related Transcripts

5.2.2.1 Sequence and Expression Analysis of Putative Phenylacetaldehyde Synthase

(VMPPAAS)

The BLASTX and BLASTP analyses (NCBI) show the deduced amino acid sequence of

VMPPAAS is 49-63% similar to other plant decarboxylases such as phenylacetaldehyde

synthases, tryptophan decarboxylases, tyrosine decarboxylases and aromatic amino acid

decarboxylases. GC-MS analysis of the scent of Vanda Mimi Palmer shows the presence

of phenylethyl acetate, which could be synthesized via the benzenoid and

phenylpropanoid pathways. Phenylacetaldehyde synthase from Petunia hybrida catalyzed

the decarboxylation of phenylalanine to phenylacetaldehyde (Kaminaga et al., 2006),

which might be the potential precursor for the production of phenylpropanoids including

phenylethanol and phenylethyl acetate in Vanda Mimi Palmer. Previously in floral scent

studies, two phenylacetaldehyde synthases have been identified from Petunia hybrida

(PhPAAS) and Rosa hybrida (RhPAAS). The PhPAAS and RhPAAS share ~50-60%

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identity with other plant decarboxylases including tyrosine decarboxylases, tryptophan

decarboxylases and aromatic amino acid decarboxylases (Kaminaga et al., 2006). Motif

prediction by Expasy tool showed a pyridoxal phosphate attachment site in the deduced

amino acid sequence of VMPPAAS and other plant decarboxylases (see figure 17).

Besides that, a conserved VHVDAAY motif has been identified in the deduced amino

acid sequence of VMPPAAS as reported in other decarboxylase proteins from plants and

bacteria by Connil et al., 2002 (see figure 17). Thus, VMPPAAS could be classified as

aromatic amino acid decarboxylase which might be involved in decarboxylation of

aromatic amino acids in Vanda Mimi Palmer especially for phenylalanine. The

involvement of VMPPAAS in decarboxylation of phenylalanine to phenylacetaldehyde

can only be confirmed by enzymatic assay (see Figure 42). Phylogenetic analysis of

VMPPAAS with other plant decarboxylases shows VMPPAAS is not clustered together

in the same group as PhPAAS and RhPAAS since Rosa hybrida and Petunia hybrida

come from different family and genera compared to Vanda Mimi Palmer. Localization

analysis by Localizome software shows no transit peptide in VMPPAAS amino acid

sequence, suggesting this protein might be localized in cytosol.

Relative expression analysis in different tissues shows that the putative

phenylacetaldehye synthase (VMPPAAS) was up-regulated in floral tissues compared to

vegetative tissues. Among the floral tissues of Vanda Mimi Palmer, petal showed the

highest expression of VMPPAAS and followed by sepal. In other scented flowers, petals

have been identified as the main source of scent emission and biosynthesis. Most of the

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Figure 42: Elucidation of Benzenoid and Phenylpropanoid Pathways of Vanda Mimi

Palmer. This elucidation is based on benzenoid and phenylpropanoid metabolism of

Petunia hybrida (Boatright et al., 2004; Pichersky and Dudareva, 2007).

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well studied fragrance-related transcripts have shown higher expression levels in petal

compared with the other floral parts including in Petunia hybrida (Verdonk et al., 2003),

Clarkia (Onagraceae) (Pichersky et al., 1994), and Rosa hybrida (Shalit et al., 2003). In

sepal, the high level of VMPPAAS expression (slightly lower than petal) might be due to

the similar structure and function of sepal and petal in orchid. This is supported by earlier

histological work on Vanda Mimi Palmer that showed the presence of many oil glands

(trichomes) in petals and sepals (Janna Ong Abdullah, unpublished data) suggesting

potential sites for fragrance biosynthesis and accumulation.

Relative expression analysis of putative VMPPAAS at different developmental stages

shows a developmentally regulated pattern. This expression study complements the result

of GC-MS analysis on the scent of Vanda Mimi Palmer at different developmental stages

(in section 4.1.1) whereby the emission of phenylethyl acetate in Vanda Mimi Palmer is

developmentally regulated. In bud stages including young and mature buds, the

expression of VMPPAAS showed a down-regulated expression compared to half-open

and fully-open flower stages. In addition, from the GC-MS analysis, no volatile

compound was detected in the bud stage. In other scented flowers including Clarkia

breweri, Anthirrhinum majus, Rosa hybrida and Petunia hybrida (Pichersky et al., 1994;

Nagegowda et al., 2008), no emission of fragrance-related compounds were detected at

the bud stage. Thus, during early stages of flower development, the floral tissues of

Vanda Mimi Palmer might not be ready for fragrance biosynthesis. In half-open flower

stage, the expression of VMPPAAS increased drastically and reached the highest level at

fully-open flower stage. At 14-day old fully-open flower, the VMPAAS expression level

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decreased drastically compared to the early fully-open flower stage. The developmentally

regulated pattern of fragrance biosynthesis in Vanda Mimi Palmer is also similar to other

fragrance-related transcripts of S-adenosyl-L-methionine: benzoic acid carboxyl methyl

transferase (BAMT) from Anthirrinum majus (Dudareva et al., 2003), geranyl

diphosphate synthase from Phalaenopsis bellina (Hsiao et al., 2008), and linalool

synthase from Antirrhinum majus (Nagegowda et al., 2008).

Relative expression analysis of VMPPAAS in the fully-open flower of Vanda Mimi

Palmer in a 24-hour cycle shows a differential expression whereby the expression is up-

regulated at night time and down-regulated during day time. The expression pattern of the

VMPPAAS in a 24-hour cycle is opposite compared to the emission of phenylethyl

acetate in a 24-hour cycle whereby the emission of phenylethylacetate compounds from

the fully-open flower was detected at high levels during day time and not detected at

night (see section 4.1.1). This might be due to VMPPAAS being not directly involved in

catalyzing the formation of the end product (phenylethyl acetate) but involved in the

formation of the main precursor for the end product. In addition, the precursor might be

stored in cells for few hours before being used for subsequent reaction and released as

volatile (phenylethyl acetate) during day time. The gene that encodes the rate-limiting

enzyme for the formation of phenylethyl acetate (not identified yet) might show high

expression during day time because phenylethyl acetate emission was detected high

during day time (see section 4.1.1). In other scented flowers such as Petunia hybrida and

Stephanotis floribunda, the expression of fragrance-related genes and emission of

volatiles were very high level during night time and very low level during the day (Pott et

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al., 2002; Boatright et al., 2004). Meanwhile, in Anthirrhinum majus, both fragrance-

related genes expression (mycrene synthase and ocimene synthase) and volatiles emission

were detected at high level during the night (Dudareva et al., 2003). In other scented

flowers including Petunia hybrida (Boatright et al., 2004), Anthirrhinum majus

(Dudareva et al., 2003; Nagegowda et al., 2008) and Stephanotis floribunda (Pott et al.,

2002), the volatiles emission pattern and expression profile of fragrance-related genes

which are involved in the formation of end product are usually similar.

5.2.2.2 Sequence and Expression Analysis of Putative 4-(cytidine 5'-diphospho)-2-C-

methyl-D-erythritol Kinase (VMPCMEK)

The deduced amino acid sequence of VMPCMEK shows 66-72% identity to 4-(cytidine

5'-diphospho)-2-C-methyl-D-erythritol kinase from other plants including Hevae

brasiliensis, Solanum lycopersicum, Catharanthus roseus and Ginkgo biloba. Besides

that, VMPCMEK shared the conserved ATP-binding site for functional activity of 4-

(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (Kim et al., 2008). In addition,

there are few other putative conserved motifs shared by VMPCMEK and other plant 4-

(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase as predicted by the Expasy tool.

The 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase has been reported to be

involved in phosphorylation of 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol into its

phosphorylated form, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol 2-phosphate in

the terpenoid pathway (Kim et al., 2008) (see Figure 41 in section 5.2.1). The subsequent

reactions in the terpenoid pathway might produce the main precursors including

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dimethylallyl diphosphate (DMAPP) and Isopentenyl diphosphate (IPP) (see Figure 41 in

section 5.2.1) for biosynthesis of monoterpenes and sesquiterpenes (Ogura and Koyama,

1998; Poulter and Rilling, 1981; McGarvey and Creteau, 1995). The Localizome

software predicted the presence of signal peptide in VMPCMEK amino acid sequence.

Thus, VMPCMEK might be localized in plastid since it is predicted to be involved in the

early step of terpenoid pathway, the methyl erythritol phosphate (MEP) pathway which

has been reported to occur in plastid (Lichtenthaler, 1999; Rohmer, 1999).

Relative expression analysis of putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-

erythritol kinase (VMPCMEK) in different tissues showed up-regulated expression in the

tested floral tissues compared to vegetative tissues. VMPCMEK is predicted to be

involved in the earlier path of terpenoid pathway which might contribute to the

biosynthesis of volatile terpenoids including linalool, ocimene and nerolidol, as detected

in the floral scent of Vanda Mimi Palmer by GC-MS analysis (see section 4.1.1). In the

bud, no fragrance compounds had been detected by GC-MS but the VMPCMEK

expression level was relatively high in bud compared to vegetative tissues. This might be

due to the involvement of terpenoid pathway for synthesis of other non-volatile

terpenoids including carotenoids, gibberelin and sterol which might be used either in

primary or secondary metabolisms (Bremly, 1997).

Analysis of VMPCMEK expression at different flower developmental stages shows a

developmentally regulated pattern in accordance to volatile emission pattern (in section

4.1.1), suggesting the terpenoids emission is developmentally regulated. Terpenoid

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pathway was also reported to be important for gibberellin biosynthesis whereby

gibberellins were reported to play an important role in plant growth and development

(Bremley, 1997). VMPCMEK might be involved in the early step of the terpenoid

pathway which contributes for bud development. Thus, during bud stage of Vanda Mimi

Palmer, VMPCMEK might play an important role for flower development while at open

flower stage (once the flower open until end of the flower life-time), VMPCMEK might

contribute for fragrance biosynthesis.

VMPCMEK shows a differential expression in a 24-hour cycle but the expression pattern

is totally different compared to VMPPAAS. This could be due to VMPCMEK and

VMPPAAS might be involved in different pathways in fragrance biosynthesis of Vanda

Mimi Palmer. Besides that, the expression of VMPCMEK and emission pattern of

terpenoids including linalool and ocimene (see Figure 6 in section 4.1.1) are also

different. At 2pm, the expression of VMPCMEK is at the lowest level while the emission

level of linalool and ocimene compound are detected at the highest level. The expression

of VMPCMEK increased gradually from 12.00am until 8.00am, and then decreased

gradually until the lowest peak at 2.00pm while the emission of terpenoids including

linalool and ocimene (see Figure 6 in section 4.1.1) were detected at very low levels as

early as 6.00am and increased gradually until the highest peak at 2.00pm. This might be

due to the fragrance compounds or their intermediates might not be directly emitted after

being synthesized and the compounds might be stored in trichomes for a few hours before

being released into the air as scent. The precursors of the final compounds might be

accumulated in cytosol or plastid before being used for the final product biosynthesis.

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Besides that, the volatiles emission might not be controlled at gene expression level, but

influenced by environment factors like heat and light. This is supported by the fact that

the emission of volatiles from the flower of Vanda Mimi Palmer were detected at very

high level in the early afternoon, a period when highest temperature usually occur in

Malaysia.

5.2.2.3 Sequence and Expression Analysis of Putative Cytochrome P450 Protein

(VMPCyP450)

BLASTX and BLASTP analysis (NCBI) shows the deduced amino acid of VMPCyP450

exhibited 39-67% similarity to other plant cytochrome p450 proteins including p450

protein from Oryza sativa, Populus trichocarpa, Ricinus communis, Capsicum annum

and also cytochrome P450 monooxygenase of Petunia hybrida, a well studied scented

flower. Cytochrome p450 proteins have been reported to be involved in modification of

monoterpene compounds into another compound such as oxidation of linalool to linalool

oxide (Hsiao et al., 2006; Dudareva et. al., 2004; Dudareva and Pichersky, 2000). Thus,

VMPCyP450 is postulated to be involved in fragrance biosynthetic pathway of Vanda

Mimi Palmer since GC-MS analysis of the scent of Vanda Mimi Palmer detected traces

of linalool oxide. Besides that, cytochrome P450s have been reported to be involved in

other fragrance biosynthetic pathways including benzenoid, phenylpropanoid and

lipoxygenase pathways (Ehlting et al., 2006). Thus, in Vanda Mimi Palmer, VMPCyP450

might be involved in fragrance biosynthesis eventhough the exact function of the protein

in fragrance biosynthetic pathway of Vanda Mimi Palmer is still far from understood.

Phylogenetic analysis of VMPCyP450 with closely related proteins from other plants

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shows VMPCyP450 is clustered together with cytochrome P450 monooxygenase of

Petunia hybrida (a well studied scented flower). Thus, VMPCyP450 might be involved in

fragrance biosynthesis like Petunia hybrida eventhough the cytochrome P450

monooxygenase is not well studied in Petunia hybrida. Localization analysis by

Localizome shows no signal peptide in the VMPCyP450 amino acid sequence. The same

analysis shows the presence of a transmembrane domain in the VMPCyP450 amino acid

sequence. Thus, VMPCyP450 could possibly be located in the plastid membrane like

cyctochrome C which is involved in photosynthesis. So, VMPCyP450 might be involved

indirectly in the terpenoid pathway of Vanda Mimi Palmer since monoterpene

biosynthesis in plant mostly occurs in plastid (Lichtenthaler, 1999; Rohmer, 1999).

Relative expression analysis of putative cytochrome P450 protein (VMPCyP450) in

different tissues showed up-regulated expression in floral tissues compared to vegetative

tissues. Among the floral tissues, the lip shows the highest expression, more than two

times higher than petal and sepal. This contrasting result compared to other putative

fragrance-related transcripts (VMPPAAS and VMPCMEK) in Vanda Mimi Palmer could

be due to the involvement of VMPCyP450 in other metabolisms besides fragrance

biosynthesis. As reported by Seidenfaden and Wood (1992), lip of orchid, which is a

modified petal, has a complicated structure. Transformation of a putative P450 gene into

Phalaenopsis flowers (orchidecae genera) showed a possibility for colour modification of

flowers compared to wild type where the anthocyanins level showed an increased (Su and

Shu, 2003). Thus, in Vanda Mimi Palmer, VMPCyP450 might also be involved in the

bright colour formation especially in the lip. In petal and sepal, VMPCyP450 might be

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involved indirectly in fragrance biosynthesis as cytochrome P450 proteins were reported

to be involved indirectly in fragrance biosynthesis including terpenoid, benzenoid and

phenylpropanoid, as well as lipoxygenase pathways as in other plants (Ehlting et al.,

2006). In Vanda Mimi Palmer, VMPCyP450 might be involved in terpenoids as well as

benzenoid and phenylpropanoid pathways because volatile fragrance compounds from

both classes were detected in the scent of Vanda Mimi Palmer (see section 4.1.1).

Real-time PCR result on VMPCyP450 expression at different developmental stages

shows a developmentally regulated pattern like other putative fragrance-related

transcripts (VMPPAAS and VMPCMEK). This result is expected since the fragrance

compounds emission detected by GC-MS (see section 4.1.1) is developmentally regulated

eventhough the specific location of VMPCyP450 in the fragrance biosynthetic pathway is

still not well understood. Expression analysis of VMPCyP450 at different time points in a

24-hour cycle shows a differential expression throughout the day. The pattern is totally

different compared to other putative fragrance-related transcripts (VMPPAAS and

VMPCMEK). This could be because VMPCyP450 is not only involved in fragrance

biosynthesis but also other metabolic functions in Vanda Mimi Palmer.

5.2.2.4 Sequence and Expression Analysis of Unknown Protein (VMPA28)

Analysis of the deduced amino acid sequence of an unknown protein transcript

(VMPA28) of Vanda Mimi Palmer shows no significant hit to any known proteins in the

Genebank database. Thus, sequence analysis with closely related proteins including

clustal W alignment and phylogenetic analysis cannot be carried out. Localization

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154

analysis by Localizome software shows VMPA28 does not have any signal peptide in its

amino acid sequence. Thus, VMPA28 might be localized in the cytosol. Motif search by

Expasy tool shows the presence of some sites including a N-glycosylation site (NTSN),

two protein kinase C phosphorylation sites (SxK, SxR) and a N-myristoylation site

(GAxxSN). The function of the VMPA28 can only be confirmed by functional study of

this protein.

Real-time PCR analysis on the expression of the VMPA28 transcript in different tissues

showed a slightly higher expression in floral tissues compared to vegetative tissues. This

could be due to the involvement of VMPA28 in both fragrance and non-fragrance

metabolisms eventhough the function of VMPA28 protein is still far from understood.

Expression analysis of VMPA28 transcript at different flowering developmental stages

shows a developmentally regulated pattern like other putative fragrance related

transcripts (VMPPAAS, VMPCMEK and VMPCyP450). The result is in accordance to

the GC-MS result on the scent of Vanda Mimi Palmer whereby the scent emission is

developmentally regulated. Meanwhile, expression analysis of VMPA28 transcript in a

24-hour cycle showed a differential expression which is totally different compared to

other putative fragrance-related transcripts selected (VMPPAAS, VMPCMEK and

VMPCyP450) for molecular characterization. Thus, the involvement of VMPA28 in

fragrance metabolism in Vanda Mimi Palmer could be different to these other putative

fragrance-related transcripts selected for molecular characterization.

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CHAPTER 6

CONCLUSION

The objectives of this study have been successfully achieved by biochemical analysis of

the scent of Vanda Mimi Palmer as well as isolation and molecular characterization of

putative fragrance-related transcripts that might be involved in the fragrance biosynthetic

pathways of this orchid hybrid. Two fragrance biosynthetic pathways have been

elucidated to play a role in biosynthesis of the fragrance which are terpenoid and also

benzenoid and phenylpropanoid pathways. In the terpenoid pathway, a putative

fragrance-related transcript, putative 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol

kinase (VMPCMEK) which was identified by differential screening of floral cDNA was

postulated to be involved in the early step of this pathway. The VMPCMEK transcript

might play an important role in the biosynthesis of monoterpenes and sesquiterpene

including linalool, ocimene, and nerolidol. Meanwhile, a putative phenylacetaldehyde

synthase (VMPPAAS) isolated from ESTs of Vanda Mimi Palmer might be involved in

the early path of benzenoid and phenylpropanoid pathway, possibly involved in the

biosynthesis of phenylethanol and phenylethyl acetate compounds which were detected in

the floral scent of this orchid. Besides that, there were two putative fragrance-related

genes encoding putative cytochrome P450 protein (VMPCyP450) and an unknown

protein (VMPA28) were also isolated and characterized at molecular level. These genes

might contribute to the fragrance of Vanda Mimi Palmer indirectly.

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Combination of GC-MS analysis of the scent of Vanda Mimi Palmer and expression

analysis of putative fragrance-related transcripts at different developmental stages have

shown that the floral scent biosynthesis and emission in Vanda Mimi Palmer is

developmentally and rhythmically regulated. The knowledge from this work is important

for preliminary understanding of fragrance characteristics and its biosynthetic pathway in

Vanda Mimi Palmer. In order to gain a deeper insight on the fragrance biosynthesis in

Vanda Mimi Palmer, future work should focus on isolation of full-length cDNA of rate-

limiting enzymes that are responsible for the biosynthesis of fragrance compounds. The

fragrance-related cDNA should be cloned into bacterial system and confirm their

involvement in fragrance biosynthetic pathway by enzymatic assays. Besides that, future

work should also focus on transformation of the fragrance-related cDNA into other plant

system including non-fragrance orchids and other flowers to increase their commercial

values.

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157

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APPENDIX A

GENERAL SOLUTIONS

TAE buffer LB broth (1 L)

242 g Tris base 10 g tryptone

57.1 ml glacial acetic acid 10 g NaCl

100 ml 0.5 M EDTA pH 8 5 g yeast extract

Adjust to pH 7.0

(For LB agar, 20g of bacteriological agar

must be added)

SM buffer TE buffer

5.8 g NaCl 10 mM Tris-HCl pH 8

2 g MgSO4.7H2O 1 mM EDTA

50 ml 1 M Tris-HCl pH 7.5

5 ml 2% (w/v) gelatin

NZY agar (1 L) Pre-hybridization buffer (100 ml)

5 g NaCl 10 ml 50 X Denhardt‟s solution

2 g MgSO4.7H2O 25 ml 20 X SSC

5 g yeast extract 2.115 ml 1 M NaH2PO4

10 g NZ amine 2.885 ml 1 M Na2HPO4

15 g agar 0.5 g SDS

Adjust to pH 7.5

NZY top agar (1 L) 20 X SSC buffer

5 g NaCl 3 M NaCl

2 g MgSO4.7H2O 0.3 M sodium citrate

5 g yeast extract

10 g NZ amine

7 g agar

Adjust to pH 7.5

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APPENDIX B

Cymbidium mosaic Virus Coat Protein Transcript Sequence

1 AATAAAGTAGCTATAGGCCCCTGGCGAGGGTTAAGTTACCACAATAATTTGAAATAATCA

61 TGGGAGAGCCCACTCCAACTCCAGCTGCCACTTACTCCGCTGCCGACCCCACTTCTGCAC

>>>>>>>>>>>>>>>>>>>>

Forward Primer (CMV-F) 5’-AGAGCCCACTCCAACTCCAG-3’

121 CCAAGTTGGCCGACCTGGCTGCCATTAAGTACTCACCTGTCACCTCCTCCATCGCCACCC

181 CCGAAGAAATCAAGGCCATAACCCAATTGTGGGTCAACAACCTTGGCCTCCCCGCCGACA

241 CAGTGGGTACCGCGGCCATTGACCTGGCCCGCGCCTACGCTGACGTCGGGGCGTCCAAGA

301 GTGCTACCCTGCTCGGTTTCTGCCCTACGAAACCTGATGTCCGTCGTGCCGCTCTTGCCG

361 CGCAGATCTTCGTGGCCAACGTCACCCCCCGCCAGTTTTGCGCTTACTACGCAAAAGTGG

421 TGTGGAATCTGATGCTGGCCACTAACGATCCGCCCGCCAACTGGGCCAAGGCTGGTTTCC

481 AGGAGGATACCCGGTTTGCTGCCTTTGACTTCTTCGACGCCGTCGATTCCACTGCCGCGC

541 TGGAGCCTGCCGAATGGCAGCGCCGCCCTACTGACCGTGAACGTGCTGCGCACTCGATCG

601 GGAAGTACGGCGCCCTTGCCCGTCAGCGTATCCAAAACGGCAACCTCATCACCAACATTG

661 CCGAGGTCACCAAGGGCCATCTTGGCTCCACCAACAGTCTCTATGCTCTGCCTGCACCCC

<<<<<<<<<<<<<<<<<<<<<

Reverse primer (CMV-R) – 5’-GCAGGCAGAGCATAGAGACTG-3’

721 CTACTGAATAACGCCAAACTTAATAAGGCGTGTGGTTTTCTAAAGTTTGTTTCCACTACT

781 GGCGTAATATATTTAGCCAGATAAATAAAAAAAAAAAAAAAAA

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APPENDIX C

Equations Used in Expression Analysis of the Putative Fragrance-related

Transcripts

Equation A (Standard curve)

y = mx + c

where y = CT values, m = slope of the standard curve, x = log input of template amount,

c = y-intercept of the standard curve line.

Equation B (PCR amplification efficiency)

E = (10 -1/slope

– 1) x 100

where E = PCR amplification efficiency, slope = m as mentioned in Equation A

Equation C (Relative quantity)

Q = E(lowest CT – sample CT)

where Q = Relative sample quantity, E = PCR efficiency

Equation D (Normalization expression)

Normalized expression level of target n = Qtarget n/ NFtarget n

where Qtarget n = relative sample quantity of target n, NFtarget n = normalization factor of target n

Equation E (Normalization Factor)

NF = (Q sample (ref 1) * Q sample (ref 2) * …………. * Q sample (ref n))1/n

Where NF = normalization factor, Q sample (ref 1) = relative quantity of reference gene 1 (endogenous control),

Q sample (ref 2) = relative quantity of reference gene 2 (endogenous control), n = number of endogenous

control genes used

Equation F (Scaled Normalized Expression)

Rescaled normalized expression target = normalized expression leveltarget n

normalized expression levelcalibrator

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APPENDIX D

GC-MS Analysis on Vanda Mimi Palmer and Vanda Tan Chay Yan

a) Data for GC-MS analysis of the scent of Vanda Mimi Palmer

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b) Data for GC-MS analysis of the volatiles emitted by Vanda Tan Chay Yan

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c) Data for GC-MS analysis of the essential oil of Vanda Mimi Palmer

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d) Data for GC-MS analysis of the essential oil of Vanda Tan Chay Yan

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APPENDIX E

Contigs and Singletons

No Clone name cDNA transcript Contig/

singletone 1 1 putative zinc finger (C2H2 type) family protein Contig 1

2 29 putative zinc finger (C2H2 type) family protein Contig 1

3 17 putative ATP binding / alanine-tRNA ligase Contig 2

4 47 putative ATP binding / alanine-tRNA ligase Contig 2

5 11 putative phospholipid transfer protein Contig 3

6 40 putative phospholipid transfer protein Contig 3

7 A44 putative phospholipid transfer protein Contig 3

8 57 putative S18.A ribosomal protein Contig 4

9 60 putative S18.A ribosomal protein Contig 4

10 72 putative enoyl-ACP-reductase Contig 5

11 82 putative enoyl-ACP-reductase Contig 5

12 2 putative elongation factor Singleton 1

13 3

putative 2,3-biphosphoglycerate independent

phosphatase

Singleton 2

14 4 putative DNA ligase Singleton 3

15 5 putative cycloartenol synthase Singleton 4

16 10 putative coated vesicle membrane like protein Singleton 5

17 24 putative vacuolar sorting protein Singleton 6

18 25

putative zinc finger (C3HC4-type RING finger) family

Singleton 7

19 27 putative protein kinase Singleton 8

20 30 putative ATP-dependent protease Singleton 9

21 31 putative esterase/ lipase/ thioesterase family protein Singleton 10

22 33 hypothetical protein Singleton 11

23 34 putative LITAF-domain-containing protein Singleton 12

24 35 putative kinesin Singleton 13

25 36 no significant hit protein Singleton 14

26 38 putative pleiotropic drug resistance like protein Singleton 15

27 42 putative flowering time control protein isoform Singleton 16

28 44 putative glycosyl hydrolase family protein Singleton 17

29 45 putative Leucine aminopeptidase Singleton 18

30 48 hypothetical protein Singleton 19

31 51 putative cytochrome P450 monooxygenase Singleton 20

32 52 Putative photosystem II core complex protein Singleton 21

33 59 no significant hit protein Singleton 22

34 61 putative abscisic stress ripening protein Singleton 24

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No Clone name cDNA transcript Contig/

singletone 35 62 putative alpha tubulin Singleton 25

36 63 putative syntaxin-like protein Singleton 26

37 66

putative KH domain-containing protein / zinc finger protein-like

Singleton 27

38 68 putative RNA binding protein Singleton 28

39 69 putative GRF protein Singleton 29

40 71

putative 4-diphosphocytidyl-2-C-methyl-D-

erythritol kinase

Singleton 30

41

73

putative dihydrolipoamide S-acetyltransferase

precursor

Singleton 31

42 74

putative Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein

Singleton 32

43 75 putative Aci-reductonedioxygenase Singleton 33

44 76 putative thioredoxin-dependent peroxidase Singleton 34

45 77 putative mitochondrial dicarboxylate carrier protein Singleton 35

46 78 putative 6-phosphogluconolactonase family protein Singleton 36

47 81 putative calmodulin-binding protein Singleton 37

48 83 hypothetical protein Singleton 38

49 85 putative jasmonate ZIM-domain protein Singleton 39

50 86 putative translation elongation factor 1 alpha Singleton 40

51 90 hypothetical protein Singleton 41

52 91 putative MADS box AP3-like protein Singleton 42

53 94 putative 40S ribosomal protein Singleton 43

54 95 putative 60S ribosomal protein Singleton 44

55 96 no significant hit protein Singleton 45

56 A28 no significant hit protein Singleton 46

57 A38 putative GRF-interacting factor Singleton 47

58 A40 putative chaperon/ heat shock protein Singleton 48

59 A46 no significant hit protein Singleton 49

60 A54 hypothetical protein Singleton 50

61 A58 putative pectate-lyase like protein Singleton 51

62 A66 beta-ketoacyl-CoA synthase Singleton 52

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APPENDIX F

BLASTX Results

Sequencing results of 62 clones up-regulated expressed in fully-open flower of Vanda

Mimi Palmer after elimination of Cymbidium mosaic Virus transcripts

No

Clone name

cDNA transcript

Classification

Score

E.value

1 1

putative zinc finger

(C2H2 type) family protein

protein with binding

function

164

5.00e-39

2 2

putative elongation

factor

protein synthesis

211

3.00e-53

3 3

putative 2,3-

biphosphoglycerate

independent phosphatase

Metabolism

291

3.00e-77

4 4

putative DNA ligase

cell cycle and DNA

processing

432

1.00e-119

5

5

putative cycloartenol

synthase

biogenesis of cellular

component

291

2.00e-77

6 10

putative coated vesicle

membrane like protein

cellular transport

325

2.00e-87

7

11

putative phospholipid

transfer protein

cellular transport

180

5.00e-44

8 17

putative ATP binding /

alanine-tRNA ligase

Transcription

289

2.00e-76

9 24

putative vascoular

sorting protein

cellular transport

448

2.00e-124

10 25

putative zinc finger

(C3HC4-type RING finger) family

protein with binding

function

258

3.00e-67

11 27

putative protein kinase

cell cycle and DNA

processing

161

2.00e-38

12 29

putative zinc finger

(C2H2 type) family

protein

protein with binding

function

135

1.00e-30

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No

Clone name

cDNA transcript

Classification

Score

E.value

13 30

putative ATP-dependent

protease

Energy

396

1.00e-108

14 VMPEST

putative esterase/ lipase/

thioesterase family

protein

Metabolism

333

1.00e-89

15 VMPA33

hypothetical protein

Unknown

106

1.00e-32

16 34

putative LITAF-domain-

containing protein

Metabolism

191

3.00e-47

17 35

putative kinesin

cell cycle and DNA

processing

141

3.00e-32

18 VMPA36

no significant hit protein

Unknown

19 38

putative pleiotropic drug

resistance like protein

Cell rescue, defence and

virulence

128

3.00e-28

20 40

putative phospholipid

transfer protein

cellular transport

180

6.00e-44

21 42

putative flowering time

control protein isoform

Development

102

5.00e-20

22 44

putative glycosyl

hydrolase family protein

Metabolism

241

1.00e-62

23 45

putative Leucine

aminopeptidase

protein synthesis

234

4.00e-60

24 47

putative ATP binding /

alanine-tRNA ligase

Transcription

289

2.00e-76

25 VMPA48

hypothetical protein

Unknown

121

3.00e-26

26 VMPCyP450

putative cytochrome

P450 monooxygenase

Metabolism

154

3.00e-36

27 52

Putative photosystem II

core complex protein

Energy

111

3.00e-23

28 57

putative S18.A ribosomal

protein

protein synthesis

254

2.00e-66

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No

Clone name

cDNA transcript

Classification

Score

E.value

29

VMP59

no-significant hit protein

unknown

30 60

putative S18.A ribosomal

protein

protein synthesis

254

2.00e-66

31 61

putative abscisic stress

ripening protein

Cell rescue, defence and

virulence

221

1.00e-56

32 62

putative alpha tubulin

cell cycle and DNA

processing

90.1

4.00e-17

33 63

putative syntaxin-like

protein

cell cycle and DNA

processing

184

5.00e-45

34 66

putative KH domain-

containing protein / zinc

finger protein-like

protein with binding

function

115

1.00e-24

35 68

putative RNA binding

protein

protein with binding

function

214

8.00e-54

36 69

putative GRF protein

Development

445

2.00e-123

37 VMPCMEK

putative 4-

diphosphocytidyl-2-C-

methyl-D-erythritol

kinase

Metabolism

169

7.00e-41

38 72

putative enoyl-ACP-reductase

Metabolism

154

6.00e-38

39 73

putative dihydrolipoamide

S-acetyltransferase

precursor

Metabolism

204

1.00e-51

40 74

putative Ubiquinol-

cytochrome c reductase

complex ubiquinone-

binding protein

Energy

123

4.00e-27

41 75

putative Aci-reductonedioxygenase

Metabolism

294

5.00e-78

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No

Clone name

cDNA transcript

Classification

Score

E.value

42 76

putative thioredoxin-

dependent peroxidase

Energy

155

1.00e-36

43

77

putative mitochondrial

dicarboxylate carrier

protein

cellular transport

138

1.00e-31

44 78

putative 6-

phosphogluconolactonas

e family protein

Metabolism

102

1.00e-20

45 81

putative calmodulin-binding protein

protein with binding function

94.4

3.00e-18

46 82

putative enoyl-[acyl-

carrier protein] reductase

Metabolism

417

4.00e-115

47 VMP83

hypothetical protein

Unknown

255

2.00e-66

48 85

putative jasmonate ZIM-

domain protein

Cell rescue, defence and

virulence

105

3.00e-21

49 86

putative translation

elongation factor 1 alpha

protein synthesis

259

8.00e-68

50 VMP90

hypothetical protein

Unknown

82.4

2.00e-14

51 91

putative MADS box

AP3-like protein

Development

183

3.00e-45

52 94

putative 40S ribosomal

protein

protein synthesis

241

2.00e-62

53 95

putative 60S ribosomal

protein

protein synthesis

176

1.00e-42

54 VMP96

no significant hit

protein

Unknown

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195

Note: The “no significant hit” proteins are the sequences which not homologous to any

sequence in NCBI database for the cut off e-value of 1e-05

and score more than 80. The

clones selected for verification by RT-PCR are highlighted and the abbreviations is only

apply for the selected clones for the verification.

No

Clone name

cDNA transcript

Classification

Score

E.value

55 VMPA28

no significant hit

protein

Unknown

56 A38

putative GRF-interacting factor

Development

117

5.00e-25

57 A40

putative chaperon/ heat

shock protein

Cell rescue, defence and

virulence

161

2.00e-38

58 A44

putative phospholipid

transfer protein

cellular transport

180

4.00e-44

59 VMPA46

no significant hit

protein

Unknown

60 VMPA54

hypothetical protein

Unknown

127

3.00e-28

61 A58

putative pectate-lyase

like protein

biogenesis of cellular

component

201

2.00e-50

62 A66

beta-ketoacyl-CoA

synthase

Metabolism

191

3.00e-47

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APPENDIX G

Expressed Sequence-Tags (ESTs) of Vanda Mimi Palmer

>1-1-T3 hypothetical protein

TTGGAGATTCTCGCTTCGCCGCACCCACAGCCGCGGCAGCATCTGCGGCGGTGCAGACCCGGATACGGTGAAATAAAA

GGTAGAATTTTGGTCAGAGGGCGGTAAGGAGAGGGAGCGGAAGGGGAGGAAGTGGAAGATGACGGGAAAAGCGAAG

CCGAAGAAGCACACGGCGAAGGAAATAGCAGCGAAGGTTGATGCAGCGACGACGAATCGCGGCGGGGGTAAGGCTGG

GCTCGCGGACCGATCGGGACTGGATAAGGGAGGGCATGCTAAATTCGAGTGCCCTCACTGCAGGATAACCGCGCCTGA

TATGAAGTCGATGCAGATCCATCATGAAGCCCGACACCCTAAGATTCCTTTCGAAGAATCGAAGCTCACCGATCTTCAT

GCCTCCAACGTTGGCGATTCGTCCAAGCCCCGCCCAGGGGTTCGGGGAAGCTTCAAAAAATAGCTTCAGCTTTGCTATT

CCGAATAGCACTGTGAGCTATTCAATAACCTTTGCTTTCCTTCTGTCATATTTAGCTTCAACTTTTACGATGCCCTAATGT

ATTTTTATGATATTTGCTCGACTGCATCTTGTCATCGCTGCTACAAGTAAGAACATGTGAGATTCCTTGTGTCATGGTTA

TCGGAGGACTTGAATTTGAACTATTTGTAGTAGTTTTCTTAAACGGTTGCTTGCTTTAAGGTAATGCGCTTCCCATAGCC

GATTA

>2-2-T3 elongation factor

CACCCTGGTCAGATTGGCAACGGCTACGCTCCCGTCCTGGACTGCCACACCTCTCACATTGCCGTCAAGTTTGCCGAAA

TCCTGACCAAGATCGACAGGCGGTCTGGCAAGGAGCTCGAGAAGGAGCCCAAGTTCCTGAAGAATGGCGATGCCGGTT

TCGTGAAGATGATTCCCACGAAGCCGATGGTGGTCGAGACCTTCTCAGAGTATCCTCCGCTGGGCCGTTTCGCCGTGAG

GGACATGAGGCAAACAGTGGCTGTTGGAGTCATCAAGAGCGTCGAGAAGAAGGATCCCAGTGGAGCCAAGGTTACCA

AATCTGCTGCCAAGAAGAAATAAAGCTGAATGGATAAACTCTTTGAGTCTTGTTTATGTTAAGTTATCTTTGTGTTGGTC

TAATCCGGTGCAGCTTTCCAGTATCAGATTCCAGATCCGGTGCTTGATATGCTGTGGCGGTGAGGTTTTGGTGTCTTTTA

AGCTTTGTTTTATGCGACTGTTTTAATTTGTTTGAGTTGAGAAGACTGCGTTTTCATTTTAACAGCTCTTTTGATTATGTT

TCTTGCACTGCTTGTTAAATTTATATTTATACATTTTTTAGGGTCTATATTATAAATTTATTTTTGTCTT

>3-3-T3 2,3 phosphoglycerate independent phosphatase

CTTTAACGTTCAACCTAAAATGAAGGCACTTGAAATTGCCGAGAAGGCAAGGGATGCTATTCTCAGTGGCAATTTTGAC

CAGGTACGAGTTAATATACCAAACGGTGACATGGTAGGCCACACTGGTGATATTGGGGCTACTACTGTGGCTTGTGAG

GCTGCAGACGTAGCTGTAAAGATGATTCTTGATGCCATTGAGCAAGTGGGTGGCATCTATGTTGTCACTGCAGATCATG

GCAATGCTGAGGACATGGTGAAAAGGGGTAAATCTGGGCAGCCACTTTTTGACAAAGAAGGGAAAGTTCAAATTCTCA

CCTCTCACACCCTTCAACCGGTTCCTATCGCCATCGGCGGCCCCGGCTTGAGTCCAGGTGTTCGTTTCCGGAGTGATGTG

CCTGATGGTGGCCTTGCCAATGTTGCTGCTACTGTGCTGAACTTACATGGTTTTGAAGCTCCCAGTGACTATGAAACCAC

CCTAATTGAAGTAGTTGAGAACTAAATATACTGTGTAATTTTCTATTTATAATATGCTAATGTGATTTTTATATTAGTTA

AGCTCATTGAAAATCTTAAAATAAGTTTACTCCATTGAACTGGAGAATGTTGTCAGGTTTATGTTGTCACAGTTGAGGG

GGCTGTGGGCAAAGTTTTGGGTTGTTTCACTATAGTTTATATTTGTGTGGAATCTCTCCTCACAATGACTTTCTTGCTTTG

AATATTTACGCCACTTTCGAGGGATGTTGTTTTTGCTTAAAGTTTCATATGTTGTATTATTGGATAGTTGGTTATGGCAA

AGACGATGATTGCTT

>4-4-T3 DNA ligase

TTTTGATATTCTCTACATTAATGGAAGGCCTCTCCTCCATGAGCAACTCAAAGTTCGCAAGGAGGAACTTTACAAATGT

TTTGTGGAGATACCCGGAGAATTTAGTTTTGCTACTGCAATAACATCTAATGATCTTGAAGAAATACAGAAGTTCTTAG

AAAACGCTGTCAGCTCCAGTTGTGAAGGGTTGATTATCAAAACTTTGGACAAAGATGCTACATATGCGCCTTCAAAACG

GTCAAACAACTGGTTAAAATTGAAAAAGGATTATATGGACAGTGTAGGAGACACTTTAGATCTGGTGCCCATTGCTGCC

TTCCATGGCCGAGGAAAACGCACAGGTGTTTATGGTTCTTTTCTTCTTGCTTGCTATGATGAACAAAATGATGAATACC

AAAGCATATGTAACATTGGTACTGGATTTTCTGAATTGCAGCTTGAAGAGAGGTCCAAAAGTCTTGGAAGTAAAGTTAT

TCCAAAACCAAAACCATACTACAGAGTAGCTGATTCAATGAATCCTGATGTCTGGTTCGAACCTGCAGAGGTTTGGGTA

GTAAAGGCAGCGGATTTGAGCATTAGTCCTGTTCATCGTGCTGCTTCTGGCATTGTGGATCCAAATAAGGGTATTTCTCT

GCGATTTCCTCGTCTACAACATGTCCGAGATGACAAAAACCCAGAACAAGCCACAACGTCTGAGCAGGTTGCTGAAAT

GTATCGTGCACAAAAGATCAATCATATGAACAATCAAGAGGAAGAGGATGACGAATGACGTGGTTTGCTGCTTGCAAT

CAAGTCTATCATTTCTTTGAAGTTGGCAGTGTAGTTTTCCTGGTAGAGTCTAATGCATTTACTTGATAAACTTCTTATTCT

ACTTTCTTGTGCTAGTACTTTGCCTATTAAATGTAAACNTGAATCTATGCTATGACGTTTCGTTTC

>5-5-T3 cycloartenol synthase/ squelene cyclase

ATTTCATTGAGAAGATACAGAAACCAGATGGTTCATGGTATGGTTCTTGGGCTGTTTGCTTCACTTATGGAATATGGTTT

GGAGTGAAAGGACTAATTGCAGCTGGAAAATCATATCAAAATAGTCCTTCTATCCAAAAAGCATGTAATTTCCTGTTAT

CTAAACAACTAGCTTCTGGTGGTTGGGGGGAGAGTTATCTGTCTTGTCAACATAAGGTGTACACAAATCTTGAAGGAGG

TCGCACTCATGCAGTAAACACAGCATGGGCCATGTTGGCTCTAATAGATGCTGGACAGGCTGAAAGAGATCCAAAGCC

ATTGAATCGAGCAGCAAAAACCTTGATTAACATGCAGCTGGAGAGCGGCGAATTCCCTCAACAGGAAATTATGGGAGT

Page 197: Vanda Mimi Palmer Thesis

197

TTTCAACAGAAACTGCATGATCAGCTATTCAGCATATCGCAACATCTTCCCAATCTGGGCGCTCGGAGAATACCGCACT

CGGGTTTTGAAGGCAAGCAATTAACATAACTCCATGTCTGAGATAAACAGCTCCTCGAACCTCATGCTTTTGTCTCTTGC

AAAAGCATTCTTGCTTGGAAAACAATAATGTGATATTTTAATGGAGGAAAATATATTAAAGATTCCATTCT

>6-10-T3coated vesicle membrane like protein

CTCAGCGGTGGGCCTTTTTTCATGTTTGCAATCGGCTGTTGGGATTAGGTTTGTGATAGACAGGGAAGAGTGCTTCTCCC

ATGAAGTTCCATATGAAGGAGACACTGTTCATGTTTCGTTTGTTGTGATTAAGTCTGAGACACCCTGGCATTACGGAAA

TGAGGGCGTGGATCTCGTGGTGAAAGATCCATCTGGCAATCAAATTCATGATTTTCGTGATAAGATAAGCGATAAATTT

GAATTTATAGTTCGCAAGAAAGGGCTTCATCGCTTTTGCTTCACCAACAAATCTCCATATCATGAAACAATAGATTTTG

ATGTTCAAGTCGCCCACTTCACATACTTTGAAGAACATGCGAAGGATGAGCATTTATCTCCTCTCCTTGAACAAATCAA

CAAGTTAGAAGACGCTCTTTACAATATTCAATTTGAGCAGCATTGGCTGGAGGCTCAGACCGAACGTCAAGCAATTGTA

AATGAAGGGATGAGCAGAAGGGCAATACACAAGGCACTTTTTGAATCTGCAGCACTGATTGGAGTCATTGTGCTACAA

GTGTATCTCCTCCGTCGCCTTTTTGAGCGAAAGCTTGGAACCTCTAGGGTTTAGCCAACTCAGGAGCCTGCAACTTCCAT

CACATGTTTCTTCTTTCTTCCAAACCTCTGACTTCATTCGTAACAAAATGAGCTTGTTATACAGGGAATCTTCATTCAAC

ACTGGCTTGTCTTTTTGGCTTGTTCCAGTGCTTCCCTGATCTAATAACCAGGTAATTATCAAATTATTCAGTTTGTTGCTT

AGTAAATGAGAATATATCTGTTTGTGGCAGC

>7-11-T3 phospholipid transfer protein

GTTATCACTCATTGTCAATGGCTCGCTCCACCGCTTTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCCGGCGTTT

TCCGCGAGGCGAGCGGGGCCATCAGTTGCGGTCTGGTGGCCTCATCCGTGTCATCGTGTATCAACTATATTCGAGGCGT

TGGCTCGCTCTCACCAGCATGTTGCAGTGGGGTGAAGAGACTCAACTCGCTGGCGCGCACAACCCCTGACCGTCAGGC

GGCATGTTCTTGCCTCAAGAGTTTCGCCAACCGTATCCCTAATCTGATCCCTTCCCGTGCTGCTGGACTTCCTAGCAGCT

GCGGAGTCAGCGTTCCATATCCCATCAGTACCTCCACCGACTGCTCCAAGGTGCACTGAGCTTCTTGGACGGGCTACAA

ATTACTGTTTTCGTATGATCTACAGAATAAAGTGGTCTCTGGGTCTAGTGAGGGTTGTTAATATCTTGTTGTTTGTGCGT

TTCTGTCTTATTTTAATTTATGTTCCTGTATTGTTATAAGGCTACACCCTTGGATGTGGTGAGTTTAATATTCCTGCT

>8-17-T3 ATP binding/ tRNA ligase

CGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGGCACGAGGGCGACATCCTCCGGCACCTCGTTTGC

AATGGTACAAGTACCAGCTCGCAGGTCCTGAACGACGAGGCTGACGACGACTATGGCGAAGCAGAGTCCAACGAAGC

TTGCCTACTTCGAGGGCATGTACGCGCTTCAGTCCACTGCCACCTTCCTCTCATTCGAGCAGGTAGATGGCCGATTGGC

GATGATCCTTGATTCAACCATCTTCCACCCTCAGGGTGGTGGGCAGCCAGCTGACAAAGGGGTCATTTGCGGTTCAGGC

TTCAGGTTCGTGGTGGAGGATGTCCGGTCCAACGATGGGGTGGTCTTTCATTTTGGATACATTGATGACTCTCAAGCTA

ACTGTGAATCCAACCTCAAAGAGAGACAAGAAGTTACCTTACAAGTTGATGCACAACGACGTGATCTCAATTCCAGGA

TTCATTCTGCTGGCCATTTATTGGATATTTGTATGCACAATGTAGGCTTATCTCACCTAAAGCCTGGAAAGGGTTATCAT

TTCCCTGACGGGCCATTTGTTGAATATATTGGAGTAATCTCCCCAGATCAACTGCAGATCAAACAAAAAGAGCTTGAAG

AAGAAGCAAATTCATTAATCAGTATTGGTGGAAAGGTTTCTGCTTCTGTTTTACCTTATGATGAAGCTGCCAGGTGGTG

CAATGGTGATCTCCCTAGTTACATTTTGAAGGGCAGCACCCCAAGAATTGTGAGGCTAGGTAACAATGCCGGATGCCCC

TGTGGAGGGACTCACGTTGCTGATATTTCAGACATAAAAAGCTTTAAGATTACCCAAATTCGATCAAAGAAAAGAATC

ACAAAGATCAGCTACAGCATCAACCCATGAGCTCTGTATTTCTCTGCAACATTGTTGATCTATTGCGATGATTAGTGAG

CTCCTATCATACCTGAATGTATCACCTAATTTTATTATGTTTTTGTATGATTAATCACAGTTGTTATTATTGGTCTATAGA

AATGTGAACTTTGCCT

>9-24-T3 Vascoular sorting protein

GGAACATTCTGCCTAGGCTGTACCTTCTTTGCACGGTCAGATCTATTTATATAAAATCAAAAGAGGCTCCTGCAAAGGA

TATCCTTAAGGATATTGTGGAAATGTGCCGGGGTGTACAACACCCTGTCCGGGGTCTCTTTCTGAGGAATTACCTCTGTC

AGATCAGTAGGGATAAGTTGCTTGATATTGGTTCAGACTATGAAGGGGATGGCGGCAGTGTTATCGATGCTGTTGAATT

TGTGCTTCAGAACTTTACAGAGATGAATAAACTGTGGGTCCGAATGCATTATCAGGGACCAGTTTGGGAAAAGGAGAA

ACGTGTAAAAGAGAGGAGTGAGCTTCGTGATCTTGTGGGAAAAAACCTCCATGTTCTTAGCCAAATAGATGGTGTTGAT

CTTGGTATGTACAAAGAAAATGTGCTTCCTAGGGTATTAGAACAGGTGGTCAATTGCAAAGATGAACTAGCCCAACATT

ATTTGATGGATTGTATAATCCAAGTGTTTCCAGATGAATATCACTTGCAAACTCTTGAATCATTATTAGGCGCATGTCCA

CAACTTCAGCCCACTGTTGATGTTATGGCAGTCCTATCTCAGCTCATGGATAGATTATCCAATTATGCAGCCTCTAGTAC

GGAGGTTTTACCAGAATTTTTGCAAGTTGAAGCTTTTGCCAAATTAAGCAGTTACATTGGAAAGGTCATTGATTCGCAG

CCTGAAATGCCAATTTTTGGTGCCATCGGCTTATACGTCTCACTTCTTACATTTACTCTTCATGTCCATCCAGATCGTCTT

GATTATGTGGATCAAGTTCTGGGAGAGTGTGTTAGAAAATATCTGGAAAATCGAGATTGAGGATAGCAAAGCATTAAA

CAAATAGTTGCTCTTTAAGTGCTCCAATGGAAAAGTACAATGACATAGAATATTGTTTTAAAGCTGCTANTATCCAGTG

TCATGAGCACCTTGNTATGCNNAAGAAATCATGCAGTGNNTATCAGAGCATATGAGATAACCGCATATC

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198

>10-25-T3 zinc finger (c3Hc4-type ring finger) family

CTCCCTTATGTTGACCACCAAAAGGCAAAGACTATAAAGAATGATTTTAATTTGCATAAAGACATGATTCGGTTGGAAG

TGGATGTCCAGAACCCGGAGGAGTATCTGGTTTCCTTTGTCTATGATGCTGTCGTGGACGGGAGGTAATCCGCTTGCGT

CACCATATACTACTTTGCTAAAGAAGGAGCCAACTGTAGTTTTTCCTCAACCGAAGAAGACATCTATAAACCCAGGAAT

TATCTTTTTGAGAAGGGAACGGCCAAAAATTTCTGTCAACCATCTGGGCATGGCATTGATTTAGGCTTCTTTGAGTTAG

AGAGTCTTTCAAAGCCAGTAAATGGAGAAGTCTTCCCACTAGTTATTTATGCCAAGGCATGCGGGCCAACTGATGGAG

ATGGCCACTCTTCTCAATCTTCTGCCACTCATGTGCAGATTACATTAGCCGTCATTGAGAAGAATAGTAAAGGAGAGTT

TGAAGCTAAAGTTATAAAGCAAATCCTATGGATTGCCGGCGAACGCTACGAGCTGCAAGAAATCTTTGGCCTGGAAAA

TTCTTCAACTGAACAGATTGATGATGGTGATGATGATATTGGTAAGGAGTGCGTAATCTGCTTGACAGAACCTAGGAGC

ATTGCAGTCTTTCCCTGCAGGCATTTGTGTATGTGTAGTGATTGTGCAAAATCTTTGAGGGTCAAATCAAATAAGTGTCC

CATTTGTCGTCAACCTGTTGAGAAACTAATGGAAATCAATGTAAATTTGAGGAGCTCTGATAGGTAAGATCGGCATCAG

TCTTCATGAAATTTGACAGAGGT

>11-27-T3 putative protein kinase

ATATTTGTTTGGCTGATTTTGCTAAGAGATGAGTTACGGCAGAGATGATCAAAATGATGGAATAGAGAAAAGGAAGTT

TATGAATTCTGAACCAAAAACAGCACCAGATTCTGTCCATCCTGGCAGGCCGTCCTTTGACTGCAACTATACTATTCCA

GTTGAGAACTTGGAGATTGATAAAAAGCTTTTGATTGATCCGAAGAATCTTTATATAGGATCCAAGATTGGTGAAGGTG

CTCATGGAAAAGTTTATGAGGGAAAATTTAGGGAACAAGTTTTAGCACTCAAAATTATCAATAGTGGCAACACGAATG

AGGAGAAAATGACAATTCAGGCTCGTTTCATTCGAGAAGTCAATATGATGTCTAGAGTGAAACATGAGAATCTTGTTA

AGTTCATTGGAGCTTGCAAGCACCCTATCATGGTTATTGCTACAGAGTTGCTACCTGGGATGTCCTT

>12-29-T3zinc finger (C2H2 type) family protein

GGAAGAAGACGGGAAAAGCGAAGCCGAAGAAGCACACGGCGAAGGAAATAGCAGCGAAGGTTGATGCAGCGACGAC

GAATCGCGGCGGGGGTAAGGCTGGGCTCGCGGACCGATCGGGACTGGATAAGGGAGGGCATGCTAAATTCGAGTGCC

CTCACTGCAGGATAACCGCGCCTGATATGAAGTCGATGCAGATCCATCATGAAGCCCGACACCCTAAGATTCCTTTCGA

AGAATCGAAGCTCACCGATCTTCATGCCTCCAACGTTGGCGATTCGTCCAAGCCCCGCCCAGGGGTTCGGGGAAGCTTC

AAAAAATAGCTTCAGCTTTGCTATTCCGAATAGCACTGTGAGCTATTCAATAACCTTTGCTTTCCTTCTGTCATATTTAG

CTTCAACTTTTACGATGCCCTAATGTATTTTTATGATATTTGCTCGACTGCATCTTGTCATCGCTGCTACAAGTAAGAAC

ATGTGAGATTCCTTGTGTCATGGTTATCGGAGGACTTGAATTTGAACTATTTGTAGTAGTTTTCTTAAACGGTTGCTTGC

TTTAAGGT

>13-30-T3 ATP dependent protease

GGATTTGGTCGATTAGGGATGATTTGCTTGTGCCGTCTTCTCCCTACTTCCCTGTGGAGGCTCAGGGCGGACAAGGACC

ACCGCCAATGGTGCAGGAGCGGTTTCAGAGCGTTATTAGCCAGCTATTCCAGCATAGAATTATACGCTGTGGTGGTGCG

GTCGATGATGACATGGCAAATATAATTGTAGCGCAGCTTCTTTATCTCGATGCAGTGGATCCTACGAAGGACATCGTCA

TGTATGTGAACTCTCCTGGAGGATCAGTGACAGCTGGTATGGCTATATTTGATACGATGAGGCATATTCGTCCTGATGT

TTCCACTGTATGTGTTGGATTAGCAGCCAGTATGGGTGCTTTCATACTAAGTTCTGGCACCAAGGGAAAACGCTACAGC

CTACCAAACTCCAGAATCATGATTCATCAGCCCCTTGGCGGCGCCCAAGGAGGACAAACTGATATAGACATTCAGGCA

AATGAGATGCTTCATCACAAGGCCAATTTGAATGGATATCTAGCTTACCACACGGGCCAGAGTTGGGAGAAAATAAAC

CAGGATACTGATCGAGACTTCTTCATGAGCGCCAAAGAGGCAAAAGATTATGGCTTAATTGATGGAGTAATAATGAAT

CCACTCAAAGCTCTTCAGCCTCTCTCAGCTTGAGATGATGAAGAAGAAGAATCAGATGAGTTCTACGACCATGATCAGA

GAAATCAGAGCTTAAGGATTGCAGTAATCTGCTCTGCTGCGATACTAAAACTTCCGACCGTTGAGAGCACTCATTGCCT

GATATAGTCAGTATTATTCGTCTTATTGTGCCTATCTAGAGTTAGAGTTAGCTCACAATGTTGCTTGAATATTTATCAAT

TAAATGAATATCAGAAAATTGCGGTTTT

>14-31-T3 esterase/lipase/thioesterase family protein

GTAAGGCGTTTCCGCGAAGAGGTCAGTTCACAGTTGGAATTCTGCGTCTCTATCGACGATGTCGTCTCCTGGAGTGTCC

GAGCAAAGGGTTGAAATTTTAAACAACTATGGAGAGAAACTTGTTGGTGTGCTGCATTTAGCAGGTTCCAAGAATCTG

GTGATCTTATGCCATGGATTTCGCTCCACAAAGGATGAGAAACTGTTACTTAACCTCATTGCTGCACTAACGAAAGAAG

GTGTGAGCGCCTTTCGCTTTGATTTTGCTGGAAATGGGGAAAGTGAAGGTGAATTTCAATATGGAAACTATCGCAAGGA

AGCTGACGACTTACGAGCTGTGGTGCTATATTTCTTAGAGCAAAATTTTAAAATTTGTGCTATTACTGGGCATAGTAAA

GGAGGAAATGTGGTGCTTCTTTATGCATCTACGCATAATGATGTCCCTCTCATCATTAATCTTTCTGGCCGTTTTGCATT

GGAAAGAGGAATTGAAGGACGCCTGGGGAAAGAATTCATGCAAAGAATAAAGAAAGATGGCTTTATTGATGTCAAGG

ATAAAACAGGAAAATTTGAATACCGGGTGACAGAAGAAAGCTTGATGGATCGTCTAACCACAGACATGCGTGCAGCAT

GCCATGCCATTGATAAGAAATGCAGGGTTTTGACAATCCATGGTTCAGCAGATGAAATTGTACCTTCAGAAGATGCCTT

TGAGTTCGACAAAGTCATACCCAACCACAAGCTTCATATCATTGAAGGTGCTGACCATTGCTATACTGCATGCCAAGCA

GAGCTGGCTTCTCTTGTTCTAGACTTCATAAAATCTGATCAGGTCGTCGATGCTACCACAGCACAAGTGATGTAAGAGT

TTTTCAAGCTCATTGTTGTTACTTTATCTTTCAATTTGTCACGCCATGGTCATTACTGTCTAGCTTGATGTTTCATTTTCTA

TATGTAATG

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199

>15-33-T3 Hypothetical protein

ATGAAACCCTAATTTTTGAGCTTTTGGATTTCAAAAGCGAGATTGAAGACAATGCCAGTGCTGTTTGGTTTCTACGTGA

CATTGCTCGTGAACAGGATGCAGAAGATAGCATGGTCCAAGAACACTCAGGAACAATAGAAGTTGCTGGCTTACAGTA

TAGAGATGCTCCTGTAATATATCAAACAGCAGTTGGTCAAATGACCATTTCCAAGGAAAGCCAAGGAATGGAGGGACA

CAAAAATGGGGGGGGGTTTAAGGGGAAATATTTCGCGTCCCAGGATTTTGCGGTGAGGGGGGCGCGTGATCACTGCAT

ATGAGCAAATTTTGGACAATTCCTTAAGTGAGAGTAGAAGCATTGAGGAAGCTGATCCAACAGTTTCTGGCAGCCTATC

TGCTGCAGAAGTCTTTAAACTATCAGCTGCAAGCTTTAAGGTACATGACTGGAATCTCTTTGGTGCTGGGGCTTGACTA

ATTATTTAATATGATTTTAAGAATTTTTGGTTATGAAAAACATTTTCATATTATTTTAATATGATTTTAAGATTTTATTTG

ACTTGTATTTTTATTTTTTAAAACTGGTGCTTAATCTGTAATTCTGTTCAACTATAATTATTTTTACAGGCGATGAAGCAA

CTGATGATGGGAATTT

>16-34-T3 LITAF domain containing protein

GTGAAACGGAAGAACTGATGGCAGCGAAGGGTTCAGGCGACGAACCTGCTCTTGGGGTGCCCTACCACTATGCATCCG

GCGGTGCCGGGGTTTCCGGATATGCCCAAGGTCCGAATCCACAGCCTGCATTCTACGTGGCGCAGCACCCGCATCAAG

CTGGGTTGATCCCGCCGAATGCGGTTTTCGGAGATCCAAAGGGCATCCCGCTCCAGCAGACGATGTTCCGGGATACCCC

CGCACCTTTCGAGTGCGTTTACTGTGGATCATCCAGTCTTACCACCATCAGATCCAAACCAAGTCTAGCTGCTCTTGTTG

GTTGCATGATGCCATTTATGCTGGGAGTATGTTTTCTTTGCCCTTCCATGGACTGCCTCTGGCACAAATATCACTATTGC

CCAAATTGTAAAGAGAAGGTTGCCTACTTTGAAAAATCAGACCCTTGTATTGTGGTGGATCCAACTAATTGGACCGAGC

CCAGCTATGCCATCCCCAGGTTGGTTTGAACTCTCTTTGAAGTTTTTGCGGATATATATATATATATATATATATATATA

TATATATATATACGGTTTTTGCTTTTGGATTTGTAAATAATGAAGTCCTCTTTGT

>17-35-T3 putative kinesin

AGGAAGAGNCCTTAATAGCAGCTCATAGAAAAGAAATTGAAAATACAATGGAGATAGTTCGGGAGGAAATGAATTTA

TTGGCGGAGGTGGACCGACCAGGAAGCCAAATTGACACGTATGTATCGCAGCTCAGCTTCCTGCTGTCGCGGAAGGCT

GCCGGGCTGGTCGGCCTACAAGCTCGCCTGGCCAGATTTCAGCACCGGCTGAAAGAGCACGAGATTCTCAGTCGCAAG

AAGGCTTTGAGGTAATAAATGATCTGCGTGCTTTTCTTTCTTTTTAATGTATCTTCTACCAGCAGAATGTCCCCTTCCCTC

TCTCTGCTTTTTCTTGCTGATTGTTATTGCCACAGATTTCTCATATTCTCATTCTTGAAGTTATACAATAGCGGCAATGCC

GTGTGATTTACCTTTATGTGTCGTGTTTGGCCTTCTTTTATTGTTGGGAGTGAGGGTGGGTTGGTCTTTCGGAATGAAAG

AAGAAGAATGTATGGAAGCCATTGTTGATAATAGTATAGTATTTATTT

>18-36-T3 hypothetical protein

ANNAGCCNTGTGCTTTTATTTCTGGGGCCTGCTTTTGTCTATCTTGTGCCTGAGGACAATGTCTGGGAGGTAGTCTTACA

GGTTCTGGTGGCCTTAGTGTGTGTCGTTGGCGGATCCGCCTCCTTCGCTGCTTCAAATTTCTTGTCAAACTTGCAGAGAT

CCAATTGAGTTTTGGTAGTCTTGTGCTTCTGACGTGGTCATTTTGGTGCAAATGTTAGCTTGTTACTTGCTCGGATTCCTT

CTGAACTCTATGTATTAGGCAATAACAAGCAAGTCAGCTTCTGGGGAGAACTATGCATGGACAGTAGGTATTTGCTAAA

TTCTTTTTAAGATTCAGGTAATATATATATATATATATATATATATATATATATATATATATAGATATGTATGTATGTATT

TATGTATGTATGTATGGAACTGTACCTTCGGAGTATCATACAATAATTAAGCTCTAAAAATTGAATGCAATAATTTTCA

GCT

>19-38-T3 pleiotropic drug resistance like protein

GTNNAAATTCCACGGCCGAGGATGCCTATATGGTGGAGATGGTATTTCTGGGCATGCCCAATTGCGTGGACACTTTATG

GATTAGTCGCTTCGCAGTTTGGTGATATAGAAGACGAGCTCGAATCTGGTGAGATTGTGTCTCAATTTGTAAAAAGTTA

CCTTGGATTCAGACATGACTTCTTGGGCGTGGTTGCGGCTGTAATCGTTGGGATTCCAGCACTCTTTGCCTTCATGTTTG

GATTTTCAATTAAAGCTCTAAACTTCCAAAGAAGATGAAGGAATTGATGAAGTCCTAAAAGATGCCCTGGTGCTAGGA

AGTTTAACATATTCATTTTGTTAATATTCTTCCACAAGAAGAATGAAAAGTTCTCAGAGGGGGTAATAATGAAGTGAAG

GATATAATCTTTCCTGACCTATATATGAACAAGAAAATATGCTCATGAATATGTTCCTCGATGTACTTAATTTGGGCATC

AGTTCAAATGTAGAGAAGTTAGGATGGATGTTGAGAGGTCTGATTTATCTTTGTCAACTATTCAAATGTAATAGTGCAA

ATATAAAACTTTACTTGGTGGATATATAATGGGGAGATATATTAACACT

>20-40-T3 phospholipid transfer protein

GTAGAGNGTTATCACTCATTGTCAATGGCTCGCTCCACCGCTTTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCC

GGCGTTTTCCGCGAGGCGAGCGGGGCCATCAGTTGCGGTCTGGTGGCCTCATCCGTGTCATCGTGTATCAACTATATTC

GAGGCGTTGGCTCGCTCTCACCAGCATGTTGCAGTGGGGTGAAGAGACTCAACTCGCTGGCGCGCACAACCCCTGACC

GTCAGGCGGCATGTTCTTGCCTCAAGAGTTTCGCCAACCGTATCCCTAATCTGATCCCTTCCCGTGCTGCTGGACTTCCT

AGCAGCTGCGGAGTCAGCGTTCCATATCCCATCAGTACCTCCACCGACTGCTCCAAGGTGCACTGAGCTTCTTGGACGG

GCTACAAATTACTGTTTTCGTATGATCTACAGAATAAAGTGGTCTCTGGGTCTAGTGAGGGTTGTTAATATCTTGTTGTT

TGTGCGTTTCTGTCTTATTTTAATTTATGTTCCTGTATTGTTATAAGGCTACACCCTTGGATGTGGTGAGTTTAATATTCC

TGCTAT

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200

>21-42-T3 flowering time control protein isoform

TATAATCTCCTTTGCTAGACCTGCAAACAGCAGAATACGATGGAGAGGCATCGCTTCGGTGACGGTGGAAAGTACTCG

AGAATGCCGTCACGGTGGCCATCAGATACCCAACCAAACCAGCACCAACGTTACCCTAGAGGCGGTGGAATAGCCGGC

GGCGGCGAAGGCTTTGGTGGCCGATACCACCCTTACCGTGGCCAGCCTGATTATTCTTCCGCCGGCGGTCAGGGTTTCC

GCGATGGTCATGGGGACTTGGGTAGTCATCAGATGCCGATGGGTGGTCAGAAGCGAGGGTTTGCGGGCAGAGGAGGTT

CTCCAGACTTTGGTGAGGGAAACAAGTTTGCCAAACTGTTCATTGGGTCGGTTCCAAAGACTGCAACTGAAGAAGATAT

TCGTCCTTTATTTGAGGAACATGGTGATGTTGTTGAAGTGGTTTTAATTAAGGATTGGCGAACAGGTCACCAACAAGGT

AGATTCAGATGTTTTCTCATGTTCGTGATACATTGAGGTTTTCTTTAACAGGTCCTTTGGGGTTTGGGGAGCAATTTATT

TATAAGTCCTCTCAGATAATGTTTTTATCTGATTAGAGTGGTTGTATTCTGTTGCAACCAGCCCATCATTATGAGGTCAG

GGTGGTATTGAGTTTCTCAAAGAAAAAGGGAAACTTGACATTGTCGTTTCTATATTTACAGCAGCCAAAATGTTGATGA

ACTGTTTACACGTGTTGATGCTTATTGATTAAAAGCTTGGTATGCAATTAAGGCATTTTTGAACTGCTTATAGCAGTGGA

AGGGAATGATGTCTTTTGAGCCTTTCTTCTCTAAAAGATCTATGGCAGTTGTTCTTATAAGCTTAGTATTTATGTGCAAC

GGTGATGAGAACTATACACATATTTTTACTTAATAAAGCTTTGCTCTTCTTGCATTTTATGCAAATACT

>22-44-T3 glycosyl hydrolase family protein 43

GAAGCACCGGCCGTGTTCAAACATAAAGGAACATACTATATGATAACTTCAGGGTGCACAGGGTGGGCGCCGAACAGG

GCAATGGCGCATGCGGCGGAGGCGATGATGGGGTTATGGGAGACGATTGGTAACCCTTGTGTCGGCGGGAACAAAATA

TTCGAACTGACCACTTTCTTTTCACAGAGCACTTTCATAGTTCCGATTTCGGGGCTGCCGGGGGTGTTCATTTTCATGGC

AGATCGGTGGAATCCGTCGGAGCTGAGAGATTCCAGGTATGTTTGGCTGCCTCTGACAGTAGGTGGAGTGGTTGATGA

ACCATTAGAATACAATTTTGGGTTTCCAGTTTGGTCTAAGGTCTCCATTTTCTGGCATAGGAGATGGAGACTTCCGAATT

GGTGAACTGCTTAACATTGAGTGTACAATATTTTGTAGATTGTTTTACATATGGTGTATAGTGTAATAAATGTTTTTGTA

GATTTT

>23-45-T3 Leucine aminopeptidase

GTAAANNNGGTGTCGAAAAGATTGTTGATTTGGCAACGCTAACTGGTGCTTGCATTGTTGCCCTTGGACCCAGCATTGC

AGGAATCTTCACTCCCAACGATGAACTAGCAAAAGAGATCACTGCTGCTTCCGTGTTAACCGGTGAGAAATTTTGGAGG

TTGCCTTTGGAGGAGAGTTATTGGGAGTCAATGAAGTCGAGTGTTGCTGATATGGCTAATAGTGGAGGCCGTCATGGCG

GCGCTATCTTAGCTGCACTTTTCCTCAAACAGTTTGTGGATGAAAAGGTCCAGTGGGTGCATATAGATGTAGCTGGTCC

AGTTTGGAATGATAAGAAGCGCGTAGCCACAGGGTTTGGTGTATCAACTTTGGTTGAATGGGTTCTCAAGAATTCTTCT

TAAATCAAATACTGAGATGCAGCAAATGGAATAGAACAAACTTTTTTCAGGTTCAGCTCATGAAAGCATTAGGAAACA

TTCTCAAATTTACTTCAAATAAAAGAACTTTGATCTGATTTTCTGTTGTCTGATATTTTATTAGTCTGCTATGTATTAGTG

TTGTCTGTTGTCATAATTTTTATGATTTGTAGCTCTGAGTGGCAGTGTAAACTAGCTGAATTTTGAATGAATTTGAAGTG

TTGTGAGGCTTTTGGCTTGTTAAATAAACAGTCAAAACTTTGTTGTGTTTGACTTGTTTATTGATGCAGTTTGCAAACAT

GAAAATTTATTTCAAAGCTTTATAAAAATTTTAAAGTC

>24-47-T3 ATP binding / alanine-tRNA ligase

GCGANNCCTCCGGCACCTCGTTTGCAATGGTACAAGTACCAGCTCGCAGGTCCTGAACGACGAGGCTGACGACGACTA

TGGCGAAGCAGAGTCCAACGAAGCTTGCCTACTTCGAGGGCATGTACGCGCTTCAGTCCACTGCCACCTTCCTCTCATT

CGAGCAGGTAGATGGCCGATTGGCGATGATCCTTGATTCAACCATCTTCCACCCTCAGGGTGGTGGGCAGCCAGCTGAC

AAAGGGGTCATTTGCGGTTCAGGCTTCAGGTTCGTGGTGGAGGATGTCCGGTCCAACGATGGGGTGGTCTTTCATTTTG

GATACATTGATGACTCTCAAGCTAACTGTGAATCCAACCTCAAAGAGAGACAAGAAGTTACCTTACAAGTTGATGCAC

AACGACGTGATCTCAATTCCAGGATTCATTCTGCTGGCCATTTATTGGATATTTGTATGCACAATGTAGGCTTATCTCAC

CTAAAGCCTGGAAAGGGTTATCATTTCCCTGACGGGCCATTTGTTGAATATATTGGAGTAATCTCCCCAGATCAACTGC

AGATCAAACAAAAAGAGCTTGAAGAAGAAGCAAATTCATTAATCAGTATTGGTGGAAAGGTTTCTGCTTCTGTTTTACC

TTATGATGAAGCTGCCAGGTGGTGCAATGGTGATCTCCCTAGTTACATTTTGAAGGGCAGCACCCCAAGAATTGTGAGG

CTAGGTAACAATGCCGGATGCCCCTGTGGAGGGACTCACGTTGCTGATATTTCAGACATAAAAAGCTTTAAGATTACCC

AAATTCGATCAAAGAAAAGAATCACAAAGATCAGCTACAGCATCAACCCATGAGCTCTGTATTTCTCTGCAACATTGTT

GATCTATTGCGATGATTAGTGAGCTCCTATCATACCTGAATGTATCACCTAATTTTATTATGTTTTTGTAATGATTAATC

ACAGTTGTTATTATGGGTCTATAGGAAAATGTGAAACTTTGCCT

>25-48-T3 hypothetical protein

NNNAGATTGAGGTGTAGTGGAATTTGCTCGGATGAACACCCTATCAGAACTGAGCTTGTGAGTTTTGCTTCCCTCTTTG

CCCCATTGCGGCCATCAGTGAAGATAAATCCTCAAGCAGCTACCAGATTCATTGAGCATTCTTTGCCTGATTTGGCACC

AGACCAGAGGAAGAGTCTCCATAATATCAGTACAGGCAAGGGTGACCGGACTCCTTTCATGACAAACAGAGCGAAGA

AGAAAACGAAATACCAATCTTTTGAGCAGCAGTCTGCGCGTGCTGCTGCGCAGGAGTTTCTTGAGAAGGCAGCAAGGG

AGCTCTTTGGCTCAAACGATTCGGACGTGAAGGGTCCATTGCAGAACCTTAAATCAGATGATGAAGATGATGAATAAA

TGCTTTGATTTATTTTTTATTTTTTTATTTATTTCTTTTACTTCAGATAAATTGTTCTCTTTTCTTCGTGAAGTATTTGGTAA

ATTATGTTTGATAAAAAACACTTTTTTTTTCTAAGAACCTTAATTTTATGGTAGT

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201

>26-51-T3 cytochrome P450-like protein

CTGAGATCATGAGAGCTAAACAAAACGGGCATCATGATGAGACAAAGCAAGACATACTATCAAGGTTCATCGAGCTCG

CCAACACCGACAAAGAGAGTGATTTCAGCACGGAAAAAGGTTTAAGAGATGTGGTGCTAAACTTTGTTATTGCAGGGA

GGGACACTACTGCTGCAACGCTCTCATGGTTTATATACATATTAGTCACACAACCTCAGGTGGCACAGAAACTCTATAT

AGAGATGAAAGAGTTTGAGGAGATCAGAGCTGAAGAAGAAAATATAAATTTGGATTTATGTAATTTGGAAGATATGGA

TTCATTCAGAAACAGATTATCAGATTTTTCGAGGCTTTTGGATTATGATTCATTAGCAAGGCTGCAATATCTGCATGCAT

GCATTACAGAGACCCTGAGGCTGTTTCCTCCTGTTCCTCAGGTTGGGTTCAAAAAAATAAAAGGGAAGATTTACATAGA

TTTACATATATTTATGTTAAATATATAAATATTT

>27-52-T3 putative photosystem II core complex proteins

CCAACCACCCTCTCTTCCATCCCTGACCTCCTCTGCCATCGCCGCTGCGGTATTCTCCTCCCTAAGCTCAGCGGACGCTG

CCCTTGCAGTGCAACAGATCGCCGACATTGCCGATGGTGACAGCCGGGGCATTGCTCTCCTAATTCCCATAGTTCCGGC

CATCGGTTGGGTTCTTTACAACATTCTCCAGCCGGCGTTGAACCAGATCAACAGGATGAGGAGTGTGAAAGGGCTTGCC

ATTGGGTTGGGGCTCGGACTGGGCCTGAACTCAGCAACCAGTGCATCAGCTGGAGAAGTGGCGGTGATTGCTGAGGCG

GCTTCTAGGGATAATAGGGGCCTACTGTTGCTAGTTCCAGTGGGTGTGGCGATTGCTTGGGTGCTGTTCAATATACTTCA

GCCAGCGTTAAACCAGATCAATAGGATGAGGGAAAGGTAAGGTACGAACGAAGTAGAGTTTGAGCCTTGCCGTTTGTG

ATTGAGTTCTTGGATGATTATAGCTTCCTTTGTATTCATGGTCATTATATTTTATCGAGGCACATTGTAGTATGCCATGAT

TATTAGATATAATTCTTTTGCTGC

>28-57-T3 S18.A ribosomal protein

AAACCCTAGCATTCGGAAAATTTTTGAAGCAGAGAAAAAATGTCTCTAATTGCGAACGAGGATTTCCAGCACATTCTTC

GTGTGCTGAACACGAACGTGGATGGGAAGAAGAAGATCATGTTTGCTCTCACCTCCATCAAGGGTATTGGTCGCCGTTT

TGCTAACATCGTCTGCAAGAAGGCCGATGTCGACATGAACAAGAGAGCTGGTGAACTCTCTGCTGCTGAACTTGAGAA

TCTCATGACTGTAGTTGCAAATCCACGCCAATTCAAAATCCCAGATTGGTTTTTAAACAGAAAGAAGGATTACAAGGAT

GGTCGGTACTCACAAGTGGTTTCAAATGCATTGGACATGAAATTGAGAGATGATCTCGAAAGATTGAAGAAGATCAGA

AATCATCGGGGCCTGCGTCATTATTGGGGGCTTCGTGTTCGTGGGCAGCACACAAAGACTACTGGGAGGAGAGGAAAG

ACTGTCGGCGTGTCGAAGAAGCGTTGATCCTCCACCTCATTCCTGCTTTGCTTCTCTACTGCATTGTTATGTTGTCTCTCC

ATTGGAGGTTGAAACTGAAATTTTGGGCGACTT

>29-59-T3 unnamed protein product

NNNNAGNGCTCGAGAACGATGCTTCCACCTCCACCCAATAGCATGAGAACGATGCCTCCACCTCCGCCGAAATTCCAG

TCGGATCATAACTTGAGAATAATGCCTCCACCTCCGAAGTTTCGATCCGTTCTTAATGGTAATTCAGAAATGAGAGCAG

CAGCTGTTCACAGAGATTCAGAGAAAGCTAGTTTAGAACGGGTTCCTGATACCCTATTGAAGCTGGTTGAGTATGGCGA

GGAAGACGAAGAGGAAGATGACATGGTTGGCTCAGCCGAAGAATCCTGTCGAAGTGATATAATGCAAAGCACGAGCT

CAAAACCTTTTTGGGCTGTATAATAAGTAGTCTTTTGAAATTTTAGTTGGCATGTCAACATGTTTAATCTCCTTCCATTG

GCTCTCAGGCCTTCTCTGATTGATGATGAGCTTTGAAGCTATGGCTCTGTTCTATTTTTCGATTTGGATGATAAATTTAGT

GAGAAAATTTGATTTAT

>30-60-T3 S18.A ribosomal protein

CGGCGGCGGGGAGTGGAAACCCTAGCATTCGGAAAATTTTTGAAGCAGAGAAAAAATGTCTCTAATTGCGAACGAGGA

TTTCCAGCACATTCTTCGTGTGCTGAACACGAACGTGGATGGGAAGAAGAAGATCATGTTTGCTCTCACCTCCATCAAG

GGTATTGGTCGCCGTTTTGCTAACATCGTCTGCAAGAAGGCCGATGTCGACATGAACAAGAGAGCTGGTGAACTCTCTG

CTGCTGAACTTGAGAATCTCATGACTGTAGTTGCAAATCCACGCCAATTCAAAATCCCAGATTGGTTTTTAAACAGAAA

GAAGGATTACAAGGATGGTCGGTACTCACAAGTGGTTTCAAATGCATTGGACATGAAATTGAGAGATGATCTCGAAAG

ATTGAAGAAGATCAGAAATCATCGGGGCCTGCGTCATTATTGGGGGCTTCGTGTTCGTGGGCAGCACACAAAGACTAC

TGGGAGGAGAGGAAAGACTGTCGGCGTGTCGAAGAAGCGTTGATCCTCCACCTCATTCCTGCTTTGCTTCTCTACTGCA

TTGTTATGTTGTCTCTCCATTGGAGGTTGAAACTGAAATTTTGGGCGACTT

>31-61-T3 abscisic stress ripening protein

GCTCGGTGCCATAGCCGCTGGTGCTTTTGCACTGCATGAGAAGCACAAGGCAGAGAAAGACCCTGAGCACGCCCATAA

GCACAAGATAGAAGAGGAAATTGCAGCAGCAGCTGCAGTTGGTGCCGGTGGTTATGCCTTCCATGAGCATCACGAGAA

GAAAGAAGCCAAGGAAGAGGAGAAAAAGCACCATCACCACCACTTTTAAAGCTTTCAACTATATCAAGACATCCATTA

CTATGTGTTTGTAATTTATATATATATATATATATATATATATATTTTTTGGG

>32-62-T3 alpha tubulin

CTCCCGCATTGACCATAAATTTGATCTCATGTATGCGAAGCGTGCTTTTGTGCATTGGTATGTTGGAGAAGGAATGGAA

GAAGGTGAGTTTTCCGAGGCTCGGGAGGATCTTGCAGCCCTCGAGAAAGACTACGAGGAAGTCGGTGCTGAGGGTGCT

GAAGATGAAGGGGAAGATCCGGATGACTATTGAGTTAGTGGGGATTCATTGAGAGTTTGGGTGTGGTTCCAGTCTTGTT

GATTTGTTTTGTTGTACTCGTGCTAGATATGCTTTCATATTGGCATATATTTCAAACCTTTGTGGTGGTGTTCTTCCATGC

GTGATTTTCCTTTTTGGATTTTTAAAAGTTTACTGGGTGTTAAATGGAATTGCTTAGATT

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>33-63-T3 syntaxin-like protein

GTANCGGCCTGATGAAGAGGTAGAACTTTGTCTTTTTTTAATTTCTGAATGATTTGAATGTTTTTAAGATCCATAGTTTC

TACCATAATTGTTGGCTCTGCAGACTATCGACCAGCTGATAGAGACAGGAAATAGCGAGCAAATTTTCAAGAAGGCAA

TACAAGAGCAAGGACGTGGCCAGATAGTGGACACCGTTGCAGAGATCCAGGAGCGTCATGATGCCATTAAAGATTTAG

AAAGGAAGCTTCTTGAGCTGCAGCAGGTATTCTTCGACATGGCGATATTGGTCGAAGCACAAGGTGACTTGCTCGATAA

CATTGAATCCCAGGTTTCAAGTGCGGTTGACCACGTTCAATCTGGAACAACAGCTCTGCAGACGGCGAAGAGACTGCA

AAAGAATTCTCGTAAGTGGATGTGCATAGCCATTATAATTCTTCTAATTATTGTCATAGTGATCGTTGTAGCTGTCATTA

AGCCTTGGAGCAAATAGTTTCCTTACAACAGACAGCTGAAGGAACTTCAGGATCGATCGATGGATCAATCAATCAATC

AGGTGATCAACTGTTTTGTATGTCATATATTTAATATTATTATTTATTTTCGCCAACTCTTTCTGCATCTGTTGATATTTTT

GTCGTATACGCTGTCGAGAGCAGCAATTTTTTTTTAATGAATTGGATACTGTTAGCGAAAAATAAATTTGTTATTATTAT

TATTATTTTTAT

>34-66-T3 KH domain-containing protein / zinc finger protein-like

AGNGANCTGCCGTTTAACTGGAGCTAGACTGTTCATACGTGAGCATGAGAGTGATCCAAATCTTAGGAACATTGAGCT

GGAAGGAACATTTGATCAGATCAAGCAGGCGACTGGGTTGGTCAGGGAACTGATTGTCAACATCAGTACTAGTGCTGC

TCCTGTGCCCGTGAAGGCAGCTGGTGGTCTTGGGGCAGGAGGTGGTGGTGGTAGTGGTGGTCCTGGGAGCAATTTTAA

GACAAAGATGTGTGACAATTTCACCAAGGGAAGCTGCACCTTTGGAGAGAGGTGCCATTTTGCTCATGGGGCGGCTGA

GCTGCGCAAGGCAACTGCTTGAAATGTTTTAACCACAACTTTCTAAGGTCATTTGTAGCAAGCGCCGGTTCGTTCGTTTT

GTAGGTGGACTCGGAACTGTTTATGAACTAGTTGATTTCTTT

>35-68-T3 putative RNA binding protein

GTTAGGATTGCAGATTCGAGCGAGCAGATTCGGGCATTGACGGAAATGAATGGTGTTTATTGTTCTTCTAGACCTATGC

GGGTTGGTCCAGCAGCTAGTAAGAGGTCTGCCGATGCACAACAGCATGACTCTGCAAAAGCTTCATTCCAAAGCTCAC

AGGGAAACCAATCAGAAAGTGACCCAAACAACACTACAATTTTTGTCGGTGGCCTGGATCCTAATGCTACGGAGGATA

TGCTGCGGCAGGTTTTTAGCCCATATGGGGAACTGGCTCATGTGAAGATACCTGTTGGAAAGCGTTGTGGCTTTGTCCA

GTTTGTTAGGAGGGCTTGTTCAGAGGAGGCTCTACTGATGCTTCAGGGAACTCAGCTTGGCGGTCAAAAGATGCGGCTT

TCATGGGGTCGTAGTCCTTCAAGCAAGCAGTCTCAGCAGCAGGAAACTAATCAGTGGAATGGAAACTACTATGGTTAT

GGCCAAAGCTATGAAGGATATGGGTATGCTCAAGCTCCTCAGGATCCGAACATGTATTCATATGGAACTTATCCAGGAT

ACGCAAATTATCAGCAACCACAGTGAGCTTTACTCGGATTGTGGCCTTTAACTTGCAAATGGTAGCAGTTTGCAAGCGG

TATCTTCAACTTCACCAGAAGACGGTCTATAGTTATGTCTGCAGGGTATTTTAACATGGATATAATTGGTTGTGCTTTCT

TCATCTTCAGAGGCTGCGTTAGTTATGCTTTTTAAGCTTCTCTTAGCTATGATTCCTGAGTGTGTTGTCTTAACGCTTGTT

ACCTTTTCTCCTGAGTTTCAATGGTTAAAGAGTATCAATTTAACTGAGTACAAGTTATGATTTATGGATTATTAATGAAT

CAATCTGGATCAATTCTAGCTTTGCTGA

>36-69-T3 GF14 protein

AAAAGAGANCGCGATCGTTGAGACGAAGATCTAGAAAAGGTTACTGGATCATTATAAAGCAGAAGTAAAGATGTCGTC

TTCTGAGTCTTCTCGTGAGGATAATGTTTATATGGCAAAGCTTGCAGAGCAAGCTGAGCGATATGAGGAAATGGTTGAG

TTCATGGAGAAGGTGGCCAAGACGGGAAGTGTGAATGAGTTGTCTGTGGAGGAGCGCAACCTCCTCTCTGTGGCCTAT

AAAAATGTTATCGGAGCTAGACGGGCCTCGTGGAGGATAATATCCTCCATTGAGCAGAAAGAGGAAGGCCGTGGCAAT

GAGGATCGTGTGACCATTATCAAGGAATATCGTGGAAAAATTGAGACCGAGCTCAGCAAGATCTGTGATGGAATCTTG

AAGCTGCTTGATTCTCATCTGATTCCCTCTGCTTCTGCTGCTGAATCAAAGGTTTTCTACCTAAAGATGAAGGGTGATTA

CCACAGGTATCTTGCTGAGTTTAAAACTGGAGCAGAGAGGAAGGAGGCTGCTGAGAGCACACTACTGGCGTACAAATC

TGCTCAGGATATTGCATTGGCGGAACTGGCCCCTACTCATCCAATAAGACTGGGGTTGGCGCTGAATTTCTCAGTTTTCT

ATTATGAAATCCTTAACTCTCCAGATCGTGCCTGCAATCTTGCAAAACAGGCCTTTGATGAGGCCATCTCTGAATTGGA

CACCCTTGGCGAAGAGTCCTACAAGGATAGCACATTGATCATGCAACTTCTCCGAGACAACTTGACTCTATGGACTTCT

GACATAACGGAGGATGCTGGGGATGAGATCAAAGAATCCTCAAAACATGAATCATGAGGAGCTGGACCTCATTTTCAT

TTCATTTTAAGTAGATATTATTGCTTTGAAAGACTTTGTTGTGTGCGTGAACTTCTTACTTTTAACTAATGAAACAACAG

CCCTGTATATGGCGACTGGATGTGAAGATGGCGTTTTTTAATGGTTATATTAAGCTTGTGGTGTGCATCATTATGCTATT

TAAGGGCTTTCGAATTACTTTTTAGTAGATGTCAGCTTGCTACTGTTT

>37-71-T3 isopentenyl monophosphate kinase

GTNAAGCGACTTCATCTTGGTATAACTAGTTCAGTTGACCCGTTGACTCTGCTAGAAAAGATCTCTCTAAATGGAATAT

CTCAAGATGTCTGCATAAATGATCTTGAACCCCCTGCATTTGATGTTTTGCCATCCTTGAAGAAGTTGAAGCAACGTGT

GCTAGCTGCAGGGCGTGGCCAGTATAGTGCTGTTTTCATGTCTGGAAGCGGAAGCACCATTGTGGGAATTGGTTCACCA

GACCCACCTCAACTTGTTTATGATGAGGATGAATACAATGATGTTTTCATATCAGAGGCTTCCTTTCTCACTCGGCAAGA

GAATCAGTGGTACGCAGAGCCAACTTCGTCCACAGGGTCTTTGAGCAGAGAAGAGCCGTCACAAACAGGAAAATAATT

ACGATAATTTTTTTACATTCTAGACCTTCTAATTTTAATTTTTCTCACATAAAATCATATTGTATTACTGTACTTATTGTT

CATGCAAGAAAGATCGATCAAGCTATCTTTCATGAATGAGCAAAATATGCAATTTTAAAAGGCACATTTACATGCTT

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>38-72-T3 enoyl-[acyl-carrier-protein] reductase (NADH)/ oxidoreductase

AAGTGATGAGTCCCGACGGCTGTCGGGGGGGCTCTTTTCCAGATTGCTATCCGANCAAAGAGAGTGATGCAGTGTTTGA

TAGTCCTGAGGATGTCCCTGATGAAATTAAGACGAACAAGCGTTATTCAGGTTCTTTATACTGGACTGTGAAGGAAGTT

GCTGAATGCGTAAAGGATGACTTTGGAAGTATAAATATTCTTGTGCATTCCCTTGCTAACCGGCCAGAGGTGACCTAGC

CTCTATTGGAGACTTCTGAACAGGGGTATCTTGCAACTTTATCTGTTTTCTTCTACTCCTTTAAATCTCTACTCGAGGGG

GGCCGTCCTATAATAAATCCATGGGTAGCTAACATATCTCTAACTTACATTGCTTCTGAGAGAATCTTTCCAGTATATGT

TGTAGCATGATTTCGGCAAAAGCTGCACTTGAGAGTGATACTCAATTACTACCTTTTAGAATCA

>39-73-T3 dihydrolipoamide S-acetyltransferase precursor

AAAAGACTCAGTTAATGACATTGTCATAAAAGCTGTAGCACTAGCGTTGAGGAATGTTCCTGAAGCAAATGCTTATTGG

AGTGATGAGAAGGGTGAGGCCATCATATCCAGCTCCATTGACATATCAATTGCGGTGGCTACAGAGAAGGGCTTAATG

ACACCAATTGTAAGGAACGCAGACGAGAAGACATTATCAACTATATCCTCAGAGGTTAAAGAATTGGCTGAGAAGGCA

CGTAATGGAAAACTTAAACCTGAGCAGTTTCAAGGTGGGACTTTTAGCATATCAAATCTGGGAATGTTCCCTGTAGACC

ACTTTTGCGCGATTATAAATCCTCCACAGGCATGCATTCTTGCTGTTGGTCGAGGTTATCAAGTGGTTGAGCCTGTCAGT

GGCAGCGGTGGAATCGAGAAGCCTGGAATGGTGACAAAGATGAGCTT

>40-74-T3 Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein QP-C

GAGGAAAGCAGAGCGAGAGGGAGAGATGGGGAAGACGCCGGTGAGGATGAAGGCAGTGGTTTATTCCCTATCGCCAT

TCCAGCAAAAGGTTATGCCGGGATTGTGGAAAGATCTTCCGACCAAGATTCACCACAAAATCTCTGATAATTGGCTAAG

CACTGTTCTTCTCCTAGGCCCGCTCATCGGAACCTACTCGTATGTTCAGCATTACAAGGAGAAGGAGAAGCTCGCGCAC

AGGTATTGAATCTGAAGTCTTGCAGAAGATGCTGAATATGGATTGCAGTATCTGAAGATTTTTTCCTGCATAAATACTG

AGACTTTTTTTGAAGTAAATTAGATTAATAACTGCATTTTGGGCACTGGTGGGAGTATTTGTTTAATTATTTGAGTAATA

ATTATCGACATAATTGAATGTGATAGATCTTCCCAGCGTTGGTGTTTTATTTTTTGGGTCATTTACATGCATACAATACA

ATTCTT

>41-75-T3 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 2 (Aci-reductonedioxygenase 2)

NTGAGCACCGCACGCCCAAGCAGTTTGTTTCTATTGACAAACTTGCAGAACTTGGTGTTCTCAGCTGGCGACTGAATGC

TAATGACTATGAAAATGATGAAGCGCTAAAAAAGATTCGTAAGGCGAGGGGCTACTCTTATATGGACATAGTTGAAGT

TTGCCCAGAGAAGCTGCCAAATTATGAAGCCAAGATCAAGAGCTTCTTTGAGGAACATTTGCACACCGATGAAGAAAT

TCGTTATTGTCTTGAAGGAAGCGGTTATTTTGATGTGAGGGATGAAAATGACCAATGGATTCGTGTAGCTGTGAAGAAA

GGGGCAATGATTGTATTGCCTGCTGGAATTTACCATCGATTTACTCTAGACACAAATGACTACATAAAGGCTTTACGAC

TTTTTGTCGGTGAGCCTGTGTGGACTCCACACAACCGACCCAATGATCATCTCCCTGCCAGAAAGGAGTACCTTGATTT

GTTGGGGAAGAAGTCAGCTGCTGGTGGCCATCAAGCCATTGAGGCTCACTGAGGTCATTGGATAAGTACATCTCTTAAC

ACCAGTCTTAGCTGCCGACTGCGATTCCTGCTTTAATCTTATATAATCTCATTTCATGAGTGGTTTTTCACGTGTGGGTTT

GATGTGATGCCAGATGTTTCTGTTGTCTTATAGAATAAAATCGGTTGCTTTATGCCATGCCAAAAGCGTGAACATGTGG

GATTTGTTTATCTTCTGTTCCTGCTGCATGCTGGCTACTTGTATGACGCTTTATGTTAAATAAGTTTGATGAGTTTGCC

>42-76-T3 thioredoxin-dependent peroxidase

GCGTGGACGAGATCCTTCTAATTAGCGTCAATGANCCATTTGTGTTGAAGGAGTGGGCGAAGAGTTACACGGACAACA

AGCACGTGAAGTTCCTCGCCGATGGCTCAGCTAACTACACGAAGGCTCTTGGCCTGGAGCTGGATCTGAGCGAGAAGG

GGCTCGGGCTGCGTTCTCGCAGGTATGCGATCCTGGTGGACGACCTCAAGGTTAAGGTGGCTAACATTGAGGAGGGTG

GAGATTTCACCATCTCTGGTGCTGATGAGATCCTCAAGTCTCTGTGATACGCCTTCTGCTGTTTGTTGCCTCTTTTTGAGA

CGCGCTCGTTTCACGTTTCTCTTTGAATAAAAGCGCTTCTTAGCTCTTCCTGTTTTGTTTTGTGTGAAAGAGAACACAGTT

CTATAAAGTTCAAGTGTTTATTTGGTTTACTCTTTAAGGTTTACCTGCAAGAAGTTTGTATTGTTGTTTGAAGTTGGTAA

AGATCTTAATTTTATGCC

>43-77-T3 putative mitochondrial dicarboxylate carrier protein

GANTCCGCCGCCGGATTAGTGGCAGCGGTGGCGTCGAATCCGGTGGATGTGGTGAAGACGAGGGTGATGAATATGAAG

GTTGAGAAGGGGGCACCGCCGCCGTATGCCGGAGCGCTGGACTGTGCGTTGAAGACGGTGAGGGCGGAGGGACCTAT

GGCGCTTTATAAAGGGTTTATTCCTACTGTTTCTCGTCAGGGGCCCTTCACCGTCGTTCTCTTCGTCACGCTGGAGCAGG

TGCGCGCGCTTCTGAAAGATTTCTAGGTTCATCTCTATGTGAGATTTGTTGGATTGATTTGTTATGATGCCGAAAATGTA

TTGCCGCTCTGGGGGCTATGAGCAGCAGAGCGGCTAGTGTTTGTTCTTCAGAAGAATCAATTAAAATCTGAGTTTTTCT

ATTATT

>44-78-T3 6-phosphogluconolactonase family protein NAANANACCCGAGTCACCGCCAGAAAGGATAACCTTTTCGATTCCAGTGATCAACTCAGCATCAAACGTCGCCATTTTG

GCTACGGGTGATGACAAGGCCATGGCTCTGCAGTTCGTTGTCGATCACAGCTCTAGTTTTGATGCCTTTTCGCTGCCCGC

AAGGTTGGTGAACCCGACCGAAGGGAAGCTGTTGTGGTTCGTGGACAAAGCGGCAGCCTCGTTCATCGACTCTGCCGA

TGAAAGCGAGCATTTTGAAATTTAGCAGCTATGTATGTGTTCTGTATGTAGATGAGTACAGTGGGTTTGATTAGTTACTT

CTTTTGTTTAGGCCCTCTTTGAGATATCTGAAGGAAGCTGTGGTTAAGATGTGAGGGTAAAGCAGTAGATGAGGAAGTG

GATTAATAAGCATAGTGCAACATTTTGGATGGAGGGCAGTAATCTGTGAGAGACATTGCTTTTATTTTAATTAGTGAAA

GAAAATTCTCAAGAGTTCTGAATTTTC

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>45-81-T3 calmodulin-binding protein

TTGAAGAGGAAGACGGCGCTGCCGTACAAGCAGAGTTTTTTAAACTGGCTCAGATTCGCTCCGGCGTTGCGTAGTTTTA

GTTCTTCGGGGGCCGGCGATAATCATTCACCCTATCGTGGGAAAACCATGGCAGGGTCGAAGTCAATGCCTCGCTTCAG

AGAAGGGAATTAATTGAGCAAAATTGAATGAATTTGGTCAAGAAAGTTAGTTAGATTTTTTTTTCTTTTTTTTTTATTTG

CTATGATAATTGATGAGTTTTAAAGTAGTTAAATTCATTTTAAGATGAGCATAATATAAATTTTAAATGATTTATCATGA

TTATGCTTATCTTAAAATAAATATAATAATTTTAAACCTATCTTCATCAAAACATTCTGACTATTTTTTCTTCATCTAATC

AGCTATGCTTGTTGTACATAAAGGTGAGTGTGTTGTGTCTAAGTGTGTAAATTTCTACTTTGAAATGAGAAAGGATGTC

T

>46-82-T3 enoyl-[acyl-carrier protein] reductase

AGATTCTTGTTGGAACATGGGTGCCTGCATTGAACATATTTGAGACTAGCTTAAGGCGTGGAAAGTTTGATGAGTCCCG

ACGGCTGTCAAATGGCTCTCTTTTCGAGATTGCTAAGGTTTATCCCCTTGATGCAGTGTTTGATAGTCCTGAGGATGTCC

CTGATGAAATTAAGACAAACAAGCGTTATTCAGGTTCTTCAAATTGGACTGTGAAGGAAGTTGCTGAATGCGTAAAGG

ATGATTTTGGAAGTATAGATATTCTTGTGCATTCCCTTGCTAACGGGCCGGAGGTGACCAAGCCTCTATTGGAGACTTC

CAGAAAGGGGTATCTTGCAGCTTTATCTGCTTCCAGCTACTCCTTTATATCTCTACTCCAGCATTTCCTTCCTATAATAA

ATCCAGGGGGAGCTACGATATCTCTAACTTACATTGCTTCTGAGAGAATCATTCCAGGATATGGTGGAGGCATGAGTTC

GGCAAAAGCTGCACTTGAGAGTGATACTAAAGTACTAGCATTTGAAGCAGGAAGGAAAAAGCAAGTTAGAGTAAATG

CAATCTCTGCTGGTCCGCTGGGAAGCCGTGCTGCAAAAGCCATTGGTTTCATTGAGAAGATGATAGAGTATTCACATGC

TAATGCGCCATTACAGAAAGAACTGCTAGCAGATGAAGTTGGTAATGCTGCGGCTTTCTTGGTGTCCCCATTAGCTTCT

GCTATCACTGGCTCCGTAGTTTATGTCGACAATGGATTAAATACAATGGGTTTGGCAATTGACAGCCCATCTCTCTCAGT

CGAGTGATAAAGCAGAAAATCTGCATAATGATTGGTTTTTTAATAACAAGTATCCCCTTCTGTTTCAATCTCCTGCGGCA

TTAATGAATAATAATTGAGTTGGTTTCTTTCCTAGTTTCATTCGAGGTCCAGTGGTTTTATACATGTAGCTATGTGATGA

TATGATGATTCCTTTCAGTGNGTACAGTGAAAATACATTTTGATG

>47-83-T3 hypothetical protein OsI_021037

GNAANAATTCAGGAAGAAGGTCCTTATGATGATCTTGATGAGGAAAAGGGCCTCTCTCGTCGTTGCCAGCTTGTGCTCG

CATTCCTTGCATTTTTTTTACTGTTTACAACATTTTGCCTTATTATCTGGGGAGCGAGCAGACAGTACAAAGCAGATGTG

GTGGTGAAGAGCTTGTCAATGGATGATTTTTATTCGGGAGAGGGTTCGGATAGCACTGGTGTTCCGACCAAGTTGATCA

CGGTGAACTGTTCTCTGAAGATCAATGTGTATAATCCAGCATTCACATTTGGTATTCATGTCACTTCTAGTCCCATTAAT

CTCAAGTACTTGGATATTGTCATTGCTACTGGTCAAATAGAGAACTACTACCAGCCCAGAAAAAGCCATAGAACCATGT

CTTTAATACTAAGGGGAGACAAGGTTCCTCTATATGGAGCTGGTGCTGCCTTACCTCTATCCAATAGTGGTGGTTCTGTT

CCATTAACTTTGGAATTTGATTTGGTTACCCGAGCTAATGTGGTGGGAAAGCTGGTTAGGGTGAAGCATCAGAAGCATG

CCTCATGCCAAATTGTAGTTGATTCTAGCAAGAACAAGGCCATAAAGTTGTCAAAAAATGCTTGTACCTATGACTAAGT

CCCTTTATCTTCATGTTTTACTAGCAGATAATCTGCTCATGATGAGGTGAAAGAAGGAAGAGTAGTTGCATGGTCTCCA

TTTGTCTATGTTTTTTTTATGTTCATTTTTGTATATTGTTGCAATAAGACAATTTGAAGATGAGTTTTGACTTGAATTTCC

TGTACTGTAGAACTTATACTTTGTTGGTAAGTGGAAGAAGGCATTTCTCTTGT

>48-104013_85_T3 jasmonate ZIM-domain protein 1

AAGAAATCGATCGATCGAGGCGGAGGATTCAACTAGTCTTTGGGATTTTCACACTCAACGATAGAGGGTATATGGCAA

AGAGGCAGGAGAAGTCGAACTTCTCCATCACCTGCGGCCTCTTGAGCCAGTACATCAAGGAAAAGGGCAGCCTTGCTG

ATCTAGGGCTTCTCGATTCTGCTCGATTAGGCAAACATGAGGCTTATCGGCCGCTGACAACCAGGAGCTTGCTCTCAGG

AGTAGGTTTCTCCATCAATGACCCTAAAGACACCAAATCCATGGAGCTTTTTCACAAGAGTATTGGTTTCCTTCCGGCC

>49-86-T3 translation elongation factor 1 alpha

GNNAAGACCTGAAACGTGGTTTTGTGGCTTCTAGTTCAAAGGATGATCCTGCTAGGGAAGCCGCCAATTTCACTTCTCA

GGTCATCATCATGAACCACCCTGGCCAGATTGGCAATGGTTATGCTCCGGTCCTTGACTGCCACACTTCCCACATTGCC

GTCAAATTTGCAGAGATCTTGACCAAGATAGACAGGCGGTCCGGCAAGGAGCTCGAGAAGGAGCCAAAATTTCTCAAG

AATGGAGATGCAGGTTTCGTGAAGATGATTCCCACCAAGCCTATGGTGGTTGAGACGTTCTCCGAGTATCCGCCGCTCG

GAAGGTTTGCTGTGAGGGACATGAGGCAGACGGTGGCGGTCGGGGTCATCAAGAGCGTGGAGAAGAAGGATCCGAGT

GGAGCGAAGGTGACCAAATCGGCTGCCAAGAAGAAATGAATTGTGCGTTGTTGTTTGAATAAGGAGGAGCGGGAATCC

ATCGAGTTGGTTTCTGGTGTTTGGATGCAGAACTGGGTGCTTGACAGACGGTGGCACTGCTCGCTTCAGTTATCTTTTTA

GTTGTTGTCTGTGTTGTTTGTTTTTCTTGTGTTGAAGGCTGTTGTACTGCTTTTCTATGGTTGTATTATTTATCGCTGCTTA

TTATGATTGTACCGTTGTTGTTGTATGAGTTTGAATTTGATGTTATTTGAGTTTTTGTGTTCT

>50-90-T3 hypothetical protein

NGCGATCAGGTTCCAAGACTTGATTTTTATTTTCCTATGGAAGTTCACAAAGATAGCAATGAACGTTTATCCATACCCA

CATTTGGCGAGTGGGATGGGAAGGTGGGTCTACCGGACTACTCGGTTGATTTTACTAAGATTCGAGAAAATCGGCGGC

AGAACAAGAGTCGGGTGAGCTTGGGGAATGAGGATGAGCTCCTACATCGTACTGGTGTAAATAGTGACCAGAATGTTG

ACGCTCTCAAGAAGACACTCCCACTTCATCAGAAGCATGTTAACTCCACAGTGGAAAGGAAGAAAAGCAGCAGATATT

TCAACTGTTGTCGTTGTTTGGGGGCCTGAAACTGTGGCTTCATCCACACATTATGTTGCACGTGGAGGTGAGGGCCTTTG

AAGCTCTTCAGCCATCCTTTTCAAATTTCTTTCACCTCCGTTTTGTCGCTCAATCAAATGTTATGGTGATTGCAAACTACT

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TGGATTAGGATTTTTGTTATGAAGAAATGCAGGATTGGAGATGTTTCTTGGTGTCGTTCTATAAAGATGTAATGTTTGGA

TTGCTTGTTAGATTCACATATTTTTATTATTATTCGATCGCATTGTTGTAGATAGATTTTATATTTTATGTACATGAAATT

TTATCTTATTTATGATTAGTAATTTCTTATTCTAATCC

>51-91-T3 MADS box AP3-like protein 17

NCNCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTTGAGCAAACTTTGGAAGAGTCTCTGAGAATTGT

TAGGCATAGAAAGTATCATGTGATCGCCACACAAACTGACACTTACAAGAAAAAGCTTAAAAGCACAAGGGAAACAT

ACCGCGCTCTAATACATGAACTGGATATGAAAGAGGAGAATCCGAACTACGGTTTTAATGTTGAGAACCACAGCAGAA

TTTATGAAAACTCAATTCCAATGGTGAATGACTGCCCTCAGATGTTTTCCTTTAGGGTTCATCCAAATCAGCCTAATCTG

CTCGGTTTGGGTTATGAATCACACGATCTTAGCCTTGCATGATCAGCAGTACTATTATAAAAGTTTATGATTTTATTGCA

TTTTTATTATATGTTTGAAACTTTAGAATTATGAGATGGGGGATCTAGTC

>52-94-T3 40S ribosomal protein S15a

TTTGAANGATGCTCTCAAGAGCATGTACAATGCTGAGAAGCGTGGGAAAAGACAAGTTAAAATCAGACCATCTTCCAA

AGTTATCATCAAGTTCCTCCTCGTCATGCAGAAGCATGGCTATATCGGTGAGTTCGAGTATGTCGATGATCATAGAGCG

GGCAAGATTGTGGTGGAATTGAATGGGCGTTTGAACAAGTGTGGTGTCATTAGCCCAAGGTTTGATGTTGGTGTTAAGG

ATATTGAGCCATGGACTGCAAGATTGCTCCCTTCGCGTCAGTTTGGGTATATTGTGCTGACAACATCAGCAGGCATCAT

GGATCATGAGGAAGCTAGGAGGAAGAATGTAGGAGGCAAGGTTCTTGGATTCTTTTACTAGTTAAAAAATGTTTTGAA

TGAATTTTGAAGATTGTTGTCTTTTAAGTGCATTTATTTGATGTTAGCCTGCATTTCTCCTTAACTTTGAATGGTGTAAGG

CTACTTGTTATCTTTGGATCTTGAGGCATATTTCCCATTTGTGTTTGCGTATTTTTTTGCTAAGGTTACCGTTGAAAATAT

TGCAGAAAATTTTATTTT

>53-104016_95_T3 60S ribosomal protein L14 (RPL14A)

CCCTTCATAGCTTCCAGCAGATCGCAGCAGCAGTCATGCCTTTCCAGAGGTACGTGGAGATCGGGAGGGTGGCACTCGT

CAACTATGGGAAGGAGTATGGGAGACTTGTTGTTATCGTCGATGTCATCGATCAGAATCGAGCCTTAGTTGATGCTCCT

GACATGGTTCGAGGACAAATGAACTTCAAGAGGCTGTCGCTGACTGATATCAAGATTGACATCCCCCGAATTCCCAAG

AAGAGCAAACTGATCGAGGCCATGCAAGCAGCTGATGTGAAGAATAGATGGGAGAAGAGCTCTTGGGGGAGGAAGCT

GATTGTGCAAAAGAAGAGAGCTGAACTGAATGATTTTGATAGATTTAAGGTCATGTTGGCTAAAATTAAGAGAGGAGA

TTCCATCCGACGTGAGCTTTCAAAGCTAAAGAAGAGTGCTTCAAAGTGAGGCTTTTGAAGAATTAATGGCCGATTCTGT

GTCCATTATTGCAGAATTTAAGTTTGAGAGTCAACCGTTAGAACAAAATGTAGTTTTGGTCGCTGTTATGAACTTAAAA

GTTTGATGTTTTGATATTTGGATTTTTTTAAAAAAATTTGTAACTGTTTGCTTTATTATTTGAATGAATTTAAGAATTGGA

TTATTATTAATTATTCCACAGTAGAAGGAGTAATTTGTGAAACTGATTTCTGTTAATGTTTGATTGCAGATTCTACAAAC

GCAATCTTCAAAATGTATTTTTGATTGTTCAATATAGATTTTAGAAATGTCTCAATTTGTTT

>54-96-T3 hypothetical protein OsJ_006705

CTTGATTTTGCTCAGAGATCTATGCCTGTTATTGGATATAGCGCTCATGCAGGTAACAGCGGGCCAATGGCTTCTGAGT

CAGTGCCTGGTTAAAATGTCCATTTCTAAGTCGCCGAATAATGCACCTCCATAATCAATCATGAAAGGACCCCCTGGGC

TAGTTTATGTATGAATGCAAAGCCTTTATCACCATCATTGTCTGCACCATCATCGTCAACATGGGAAGGAGGTTAGCTTT

TGTTGAAGTTTAGGCGAAATTGCTCATGCCTTAACGAGCACATATCAGTTTTCGCCTTCTCTCATCTCTGAATTTGGTCT

CTTCTAACATGTAAACTTTAGAAACAGTGGGGAGTAGCCGGGAGGTTTCTTCTTCCGGGGAGCTTTATTTTGTTCGCTCT

GCATGGGAACTCCAGTAAATTTATTAGTGTCCTGGACTATACAGGTTTGATCCTGGAGATCGATCGTTTCTGCAGTTTTA

AAACTTGAAACTATCAAAAAGGTACTTTTATGCGTGGGCTTGGTGGTGTAATTATTCTGTTAGAATCTATATTTAGAGG

CTTC

>55-28 hypothetical protein

AAATATCCCGCAACCTGTCCCACCTTTTCTCCCCTTTTCGTCTCTTTTTCCCTGTTTCTCTTTCTCATCCGATTGATGGATC

TTAGGGCGGCGATAGTCGCCGCCGCCGGCGAGCGTTGGACGGAGGAGCGGCACTCCCGCTTCCTCAACTCGATCGAAA

GTACTTTCGTCCATCAAATGCTCGGCATCCATCCCGACGGCGATAACCTCCGCCGATGCGCGGCGAGGCTCGACCGTCG

TGTTCCCGATTGCATCGCCGGTAAAGAGTCTGCGAAGAGTTCTCAGATGCGATCGCCGGATAGGAGGCCTGCTGCCATT

ACTGCGGGCGCCAACACCTCTAATTGTACACGGAAACGATCACTGCGGCGATATGATGCGTCGCTAGACCAGGTGGTG

CCGGAGTTCAAAAATAAGAACGTCGGCGAGGATGCATCC

>56-38 GRF interacting factor

CTCCTCACTTGCCCGCCGCCATTCTTGTCGATTACCCACTGTTCCCTCAAAGATCCCCTTTATTCGCTTAACCCACCACC

AAAGCTGAATTTCAGAGTCCAGGACTCAAAAAGCCGAAATCTTTCCGTGTAGAAAATACGACAAGCCAACACAGGAAT

GCAGCAGCCCTCGCATCCGATGTCCCAAATTTCTCCGGGCAACATTACCACAGAGCAGATTCAGAAGTATTTGGATGAA

AACAAGCAGCTGATTTTGGCAATTTTGGATAATCAGAACTTGGGCAAACTTGCTGAATGTGCTCAGTACCAAGCCCAGC

TCCAAAAGAATCTGCTTTATCTCGCCGCCATAGCTGATGCACAGCCTCCAACTCCTTCAGTCCGTCCTCAGGTTTTCTCT

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GCACAATTTTCAAGCTTATCCTCATATATCTGGTTGTGCATAGAAGTAGAAATTTGTTAATGCTGACAGTTTGAGGAGC

GAGCTCATGGAACGAAGACTATTCAAAATTTGGGTTTGTTTATGGCTCGGTGACTATCATGCTTACTTGAAACAACTTT

GGTTTACCTCGGTATAGATCAAATAATTTTGCTTGAGATGAAATCTAAATAAAGGGTTGGTTTGTTCTATATATAATGA

AGTCTTTTACCTAGCACACTATTAGATATGGTTGTGAAGAGCTTCTTATAATAAAAAATTTTGGGAAAGC

>57-40 chaperon heatshock protein

GCAAGCTGAAGATGGATGCCAGAGTTTTCAGATTGGAGAACCCCCTCTTCTACGCACTGCAAAATTTGGTGGACCTGCC

GGAAGAAATGGAGAAAGCCTTCAACGCTCCGACGAAGAAATATGTACGCGACGCGCGGGCGATTGCTTCCACGCCGGC

AGACGTGATTGAGAAGCCGACGGCCTACGAGTTCGTCCTAGATATGCCGGGAGTGAAATCTGGCGATATAAAGGTCCA

GGTAGAGGAGGGTAACGTGCTCGCCATTACAGGGGAGCGGCGCCGGGAGGACGAAGACAAGGAAGGCGGCGTAAGTT

ACCTTCGATTGGAACGACGGGTAGGGCAATTTCTGCGCAAGTTCTCTCTCCCGGATGACTCCAATCTCGAAGCGATAAC

CGCTGTCAGCCGTGATGGTGTTCTGACAGTTACCGTGCAGAAGCTGCCGCCGCCCGAGCCTAAGAAACCCAAGACAAT

CGAGGTCAAGATTGCGTGAATTGATCTGTTCGTTTTGAATTTTTAGGGTTTGCTTTACTGTCATTATGAAGTATGTTGAG

GTTGTGTCTAAGGATTGGATCCTTGTGATCTGCTTTGTTTCCAATCGCTTTTTGATATATCTATAATACTATCAGTAAAG

ATTTTGTGGCTTTGGG

>58-44 phospholipid transfer protein

CTCGCTCCACCGCTTGTGTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCCGGCGTTTTCCGCGAGGCGAGCGGG

ACCGTCACTTGCGGCCAGGTGGCCTCAAACGTGGCACAGTGCATCGGCTATATTCGAGGCGCTGGCTCGCTCACACCAG

CATGTTGCAGTGGGGTGAGGAAACTCAACGGGCTGGCGAGCACCACCCCTGACCGTCAGACGACATGTTCTTGCCTCA

AGACTCTCGCCGGCAGTATCAAGGATCTGAACCGTGGCCTTGCTGCTGGACTTCCTGGAAAGTGCGGCGTCAACGTTCC

ATATCCCATCAGCACCTCCACCGACTGCTCCAAGGTGCGCTGAGCTCCAAGGACGGGCTACTCATCGAGCACTGCTTTG

GTTCGATGATCTGCAGAATAAAGTGGAGACTGGATCTATTGAAGCTATTTTATGTATTGTTGTTTGTGGGTTCTGTCTTT

CGATTTTAGTTTATTGTTTTGCAAGAGGGTGCACCCTTGGTTGTGGTGTGCTGGATATTGTTGCTGTAATATTATCACTTT

TTCGACTAATATGAGTTGCAGCATATTATAATT

>59-46 hypothetical protein

GGAGAAGCCTAAGGAGCCGGAGAAGGAGAAGCCTAAACAGCCTGAGCCGCCTCAAGGGGGCCAGCCGGTCTGGCCGC

CCGGTCCACACATGCCATGCTGCTGCTGGAGACCATGCTATGAGCAGTACTACGGCGGCTGTCGGTGCTGCTCCTGTGG

GATGTTCTACGGGTGGACGGGGCCGCCATTGCCACCGGCTATGGGCACTCCGTCAACATCCACGTGTCAGTTCTTCTGC

GAGGAGGATCCTTCTACATGTACCATTATGTAATAATATAGTCCCTTTCGTTCAAATTATGAGCCATTAATAGAGATGA

TAAATAAAGCTGTGCTCCTCTCTATAGTGCATTGAGTTATAATAACTCAAGAGATATATGTCAGTTTTATAATATTTTGT

CTTCTTTATTTGAATATGTAATTTTGTTTTCATCATGGTGAAAGATATATGTCAACTTTGCAATATTTTGTTTTCTTT

>60-54 hypothetical protein

CTGAGAAGCAGTCCTCGTACACGTACTGGGTGAGGGAGACGAAGGACGACGCGGCGCCGCTGCAGGTGCCTCGGAAG

CTCACCCCGGAGGACGTTTCTCAGCAATCCCAGCCTAACTTGTTGGGATCCGTATGGAATCAGGCCGGAACAtGGGAGG

AAAAAAATCTAAATTCATGGGCAAATAGTAGAATTAAGGAGCTTCTCAGTTCATTGTCCTTGGAGTTTTCTAATGGAAA

AGCAGCGGTTTATGAAGTTACCAAATGTTCAGGGGATGCATTTTTGATCACGGTGCGGAAC

>61-58 pectate lyase protein

GCGATTATGATGATGGTTTGATTGATATCaCaCgAGAGAGCACTGACaTCaCTATCTCgAGATGCCACTTTGCAATGCATG

ATAAAACAATGCTTATTGGGGCTGATAGCAGCCATATTACTGATAGATGTATCCGGGTGACAATACATCACTGTTTTTT

TGATGGAACAAGACAGAGACACCCTCGTGTTAGATTTGGGAGAGTTCACCTCTACAATAATTATACAAGAAATTGGGG

TATATATGCAGTATGCGCTAGTGTGGAAGCaCAGGTTCTCTCTCAGTGCAATATATATGAAGCCGGAGAG

>62-66 hypothetical protein

CCCACCCCTTCCCTATCTTCCATGATAATCAACCACTACAAGCTTAGGGGAAACATCATCAGCTACAACCTTGGTGGTA

TGGGATGTAGTGCTGGACTCATCTCCATAGATCTTGCAAATCGTCTTCTTCAAGTTCATCCCAACTCCTATGCTTTGGTT

ATCAGCATGGAGAACATTACTCTCAATTGGTACTTTGGAAATACCCGTTCCATGCTCGTTTCGAATTGCTTGTTTCGAAT

GGGCGGCGCAGCTATTCTCCTATCCAACAGACGGTCAGATCGCCGTCGATCCAAGTATCAGTTAGTCCATACGGTTCGC

ACGCATAAGGGCGCCGACGAAAAATGTTTTGCTTGTGTGACTCAACAAGA

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APPENDIX H

Real-time RT-PCR Data

1) PCR Efficiency for Putative Fragrance-related cDNAs and Housekeeping Genes

(D)

(C)

(B)

(A)

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Notes: PCR efficiency of putative fragrance-related cDNA and housekeeping gene

cDNA transcripts for real-time RT-PCR analysis. (A) putative actin (B) putative

cyclophylin (C) putative alpha tubulin (D) VMPPAAS (E) VMPCMEK (F) VMPCyP50

and VMPA28

(F)

(E)

(G)

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2) Expression Analyses of Putative Fragrance-related cDNA Transcripts and

Reference Genes Transcripts

a) Expression Analyses in Different Tissues

VMPPAAS

Tissues Calibrator Relative Quantity Standard Error

bud x 1 0.238

fully-open flower 8333 168.76

petal 20256 888.56

sepal 15879 487.98

lip 4439 567.47

leaf 0.5 0.007

root 0.3 0.0065

shoot 0.3 0.0054

stalk 0.4 0.076

VMPCMEK

Tissues Calibrator Relative Quantity Standard Error

bud x 1 0.16757

fully-open flower 1.63 0.20777

petal 1.763 0.2645

sepal 1.81 0.1436

lip 0.609 0.02515

leaf 0.009 0.00095

root 0.015 0.0042

shoot 0.012 0.00155

stalk 0.012 0.00121

VMPCyP450

Tissues Calibrator Relative Quantity Standard Error

bud x 1 0.17897

fully-open flower 3.094 0.27044

petal 1.12 0.17177

sepal 1.912 0.18797

lip 5.497 0.30796

leaf 0.001 0.00008

root 0.003 0.00047

shoot 0.001 0.00005

stalk 0.002 0.00016

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VMPA28

Tissues Calibrator Relative Quantity Standard Error

bud x 1 0.10546

fully-open flower 1.17 0.09434

petal 1.266 0.10744

sepal 1.352 0.04426

lip 1.054 0.06094

leaf 0.586 0.06464

root 0.62 0.04724

shoot 0.482 0.04496

stalk 0.644 0.06778

b) Expression Analyses at Different Flower Developmental Stages

VMPPAAS

Stages Calibrator Relative Quantity Standard Error

young bud (green) x 1 0.045

mature bud (red) 8.711 0.578

half-open flower 49277.86 4691.799

fully-open flower 83509.328 6075.784

14-days old fully-open flower 7617.29 1163.816

VMPCMEK

Stages Calibrator Relative Quantity Standard Error

young bud (green) x 1 0.10003

mature bud (red) 1.508 0.21186

half-open flower 2.273 0.22069

fully-open flower 1.822 0.23132

14-days old fully-open flower 0.123 0.10549

VMPCyP450

Stages Calibrator Relative Quantity Standard Error

young bud (green) x 1 0.10579

mature bud (red) 2.734 0.27015

half-open flower 3.344 0.49873

fully-open flower 3.041 0.22863

14-days old fully-open flower 2.094 0.29847

VMPA28

Stages Calibrator Relative Quantity Standard Error

young bud (green) x 1 0.25742

mature bud (red) 1.98 0.24924

half-open flower 2.5 0.34808

fully-open flower 3.5 0.26529

14-days old fully-open flower 2.8 0.28955

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c) Expression Analyses at Different Time Points in a 24-hour Cycle

VMPPAAS

Time Calibrator Relative Quantity Standard Error

12am x 1 0.09914

2am 0.601 0.01281

4am 0.474 0.03276

6am 0.328 0.13436

8am 0.023 0.05484

10am 0.012 0.00981

12pm 0.011 0.0071

2pm 0.001 0.00021

4pm 0.001 0.00021

6pm 0.154 0.04827

8pm 1.404 0.01737

10pm 3.361 0.29116

VMPCMEK

Time Calibrator Relative Quantity Standard Error

12am x 1 0.18511

2am 1.87 0.22595

4am 1.954 0.27654

6am 2.437 0.30942

8am 2.613 0.34981

10am 2.179 0.33652

12pm 1.424 0.25509

2pm 0.857 0.09788

4pm 1.13 0.12365

6pm 1.15 0.17864

8pm 1.342 0.18711

10pm 1.778 0.25471

VMPCyP450

Time Calibrator Relative Quantity Standard Error

12am x 1 0.18057

2am 0.158 0.02676

4am 0.347 0.06629

6am 0.654 0.06927

8am 0.813 0.10608

10am 0.819 0.09146

12pm 0.445 0.07637

2pm 1.56 0.16879

4pm 2.378 0.11831

6pm 3.808 0.23655

8pm 4.586 0.33069

10pm 6.367 0.10896

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VMPA28

Time Calibrator Relative Quantity Standard Error

12am x 1 0.2131

2am 1.562 0.17269

4am 2.013 0.2717

6am 2.196 0.27402

8am 2.981 0.39576

10am 1.376 0.02264

12pm 0.635 0.18099

2pm 1.879 0.2764

4pm 4.188 0.2486

6pm 2.18 0.2475

8pm 0 0

10pm 0 0

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3) Melting Curve analysis

(a) VMPPAAS

(b) VMPCMEK

(c) VMPCyP450

(d) VMPA28

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(e) Actin (Reference gene)

(f) Cyclophilin (Reference gene)

(g) Tubulin (Reference gene)

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BIODATA OF THE AUTHOR

Mohd Hairul Ab. Rahim was born on 18 September 1985 in Telok Mas, Melaka. He went

to SK Telok Mas primary school from 1992-1997. He continued his secondary education

in SMK (A) Sultan Muhammad, Melaka from 1998-2000, and then SM(A)P Kajang from

2001-2002 to obtain his Sijil Pelajaran Malaysia (SPM). His pre-university education was

completed in Malacca Matriculation College. In July 2004, he entered Universiti Putra

Malaysia to begin his undergraduate study and completed his Bachelor of Science degree

in Biotechnology with a CGPA of 3.372. He continued his Master degree in the field of

Plant Biotechnology at Universiti Putra Malaysia in 2007.

He was active in extracurricular activities throughout his undergraduate studies. He was a

member of the Students‟ Representative Council of Universiti Putra Malaysia for

2005/2006 session and also an Exco of BioMix Society, an undergaduate society of

Faculty of Biotechnology and Biomolecular Sciences UPM for 2004/2005 session.

Besides that, he was involved in many students‟ programs including „Sambutan Hari As-

syura Universiti Putra Malaysia 2006 organized by the Students‟ Representative Council

of Universiti Putra Malaysia (The Director of the programme), commitee members of

„Faculty Night 2005 and 2006‟ organized by BioMIX Society and a facilitator for

„Minggu Perkasa Putra 2005‟ (orientation week for incoming new students of Universiti

Putra Malaysia). During his undergraduate studies, he was selected to present a poster

presentation at 18th Intervarsity Biochemistry Seminar for his final year project. He

received the Public Service Department (JPA) scholarship for his undergraduate studies.

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His interest on molecular biology and plant biotechnology started in 2006 during his

practical training at Molecular Biology Laboratory, Makmal Biaklon, Felda Agricultural

Services under the supervision of Dr. Sharifah Shahrul Rabiah Syed Alwee. During his

postgraduate studies, he had presented in some conferences and seminars such as Asia

Pacific Conference on Plant Tissue Culture and Agrobiotechnology (APaCPA) 2007

(poster presentation), 5th Malaysian International conference on essential oils, fragrance

and flavour Materials (MICEOFF5) (oral presentation), 18th

Scientific Meeting of the

Malaysian Society for Molecular Biology and Biotechnology (MSMBB) (Poster

presentation) and 2009 Plant Biotechnology Postgraduate Symposium (Oral

presentation). He won the first prize for best oral presenter for the Molecular Biology,

Agriculture and Physiology category in the 2009 Plant Biotechnology Postgraduate

Symposium. Besides that, he was also a recipient for a research grant from Malaysia

Toray Science Foundation (MTSF) 2008 for the screening of fragrance-related cDNAs

from Vanda Mimi Palmer. During his postgraduate studies, he was financially supported

by the Graduate Research Fellowship (GRF), Universiti Putra Malaysia.

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LIST OF PUBLICATIONS

Terpenoid, Benzenoid and Phenylpropanoid Compounds in the Floral Scent of Vanda

Mimi Palmer. Mohd-Hairul, A.R., Namasivayam, P., Cheng Lian G.E. and Abdullah,

J.O. (2010). Journal of Plant Biology 53: 358-366.

Putative Phenylacetaldehyde Synthase Transcript of Vanda Mimi Palmer: Sequence and

Expression Analysis. Mohd-Hairul, A.R., Chan, W.S., Namasivayam, P., Cheng Lian

G.E. and Abdullah, J.O. (2010) International Journal of Botany. Published online on

September 2010. http://scialert.net/abstract/?doi=ijb.0000.20116.20116#

Screening, Isolation and Molecular Studies of Putative Fragrance-related Transcripts of

Vanda Mimi Palmer. Mohd-Hairul, A.R., Namasivayam, P., Abdullah, J.O. and Cheng

Lian G.E. (2010). Status: Submitted


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