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Isolation and characterization of oilpalm constitutive promoter derived fromubiquitin extension protein (uep1) gene

Subhi Siti Masura1, Ghulam Kadir Ahmad Parveez1 and Ismanizan Ismail2

1Advanced Biotechnology and Breeding Centre (ABBC), Biological Research Division, Malaysian Palm Oil Board (MPOB), P.O. Box 10620, 50720 Kuala Lumpur,Malaysia2 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

The ubiquitin extension protein (uep1) gene was identified as a constitutively expressed gene in oil palm.

We have isolated and characterized the 50 region of the oil palm uep1 gene, which contains an 828 bp

sequence upstream of the uep1 translational start site. Construction of a pUEP1 transformation vector,

which contains gusA reporter gene under the control of uep1 promoter, was carried out for functional

analysis of the promoter through transient expression studies. It was found that the 50 region of uep1

functions as a constitutive promoter in oil palm and could drive GUS expression in all tissues tested,

including embryogenic calli, embryoid, immature embryo, young leaflet from mature palm, green leaf,

mesocarp and meristematic tissues (shoot tip). This promoter could also be used in dicot systems as it was

demonstrated to be capable of driving gusA gene expression in tobacco.

IntroductionOil palm has been identified as a renewable factory for the large-

scale production of plant oil-derived chemicals in the future [1].

Introduction of useful genes through genetic engineering will

enhance the oil palm yield and increase its agronomic traits.

Constitutive expression of transgenes in the whole plant is

required for certain traits such as vaccine and polymer production

[2,3], disease resistance plant [4,5], tolerance to abiotic stresses

[6,7] and herbicide and antibiotic resistance [8]. To meet the above

requirement, the use of a strong constitutive promoter capable of

driving a high expression of transgenes in most tissues is essential.

The promoter is very important for producing the above traits as

well as providing a good understanding toward regulation of

transgene in transgenic plant.

A prominent example of a strong promoter that is commonly

used for directing constitutive expression in transgenic plants is

the CaMV35S promoter, which originated from the cauliflower

mosaic virus [9]. Another promoter that is commonly used to

drive a high transgene expression in monocots is maize poly-

ubiquitin promoter. To date, the polyubiquitin promoters have

Corresponding author: Parveez, G.K.A. (parveez@mpob.gov.my)

1871-6784/$ - see front matter � 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2010.01.337

been isolated from several monocot and dicot plants such as

sunflower [10], tobacco [11], rice [12] and maize [13]. Many

studies have indicated that the constitutive nature of ubiquitin

genes accounts for the ability of their promoters to constitu-

tively drive reporter gene expression in transformed cells and

plants. The constitutive status of these genes is due to the

presence of important sequences or motifs in the promoter

region. In the maize ubi1 promoter region, two overlapping heat

shock sequences were found at positions �214 and �204. The

promoter did not contain GC boxes, but the sequence 50-

CACGGCA-30 (function unknown) occurred 4 times, at positions

�236, �122, �96 and �91 [13]. Additionally, most studies

indicated that constitutive promoters might contain multiple

cis-acting elements, each of which interacts individually with

cell- or tissue-specific trans-acting factors [14,15]. The combined

activities of individual cis-acting elements confer constitutive

gene expression in most tissue types. Furthermore, this synergy

of multiple cis-acting elements has been found in CaMV35S [14]

and rice actin 1 [15] constitutive promoters.

A promoter derived from another ubiquitin family, the ubiqui-

tin extension protein (uep) gene, has been isolated from yeast [16]

and several plants, including maize [17], tomato [18], barley [19],

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potato [20], and Arabidopsis [21]. In general, this gene exists in two

isoforms in higher plants [21] and in other eukaryotic organisms

[16]. The single ubiquitin coding unit is translationally fused to a

coding region for either 76–81-amino-acid or a 52-amino-acid

ribosome-associated polypeptide. Promoters for two different ubi-

quitin extension protein isoforms have been isolated from Arabi-

dopsis and tested in heterologous systems. These studies

demonstrated that b-glucuronidase (GUS) expression driven by

these promoters was constitutive in transgenic tobacco [21]. Simi-

lar results have also been acquired in potato [20]. However, the

efficiency of promoters derived from this class of ubiquitin gene

has not been tested in monocot systems. In the present study, an

oil palm ubiquitin extension protein gene was constitutively

expressed in all oil palm tissues tested. To further investigate

the constitutive nature of the ubiquitin gene, its 50 region was

isolated and characterized. These outcomes of this work will

facilitate genetic engineering program of oil palm. Additionally,

such promoters may prove effective for the production of trans-

genic plants with agronomically beneficial traits in other monocot

and dicot systems.

Materials and methodsReverse northern analysesReverse northern analyses were performed according to the man-

ufacturer’s instructions (Bio-Dot1 Microfiltration Apparatus,

BIO-RAD). Wells of dot blots were rinsed with 300 ml 2� SSC

(3 M NaCl, 300 mM tri-sodium citrate, pH 7.0). About 200 mg of

PCR derived fragment was added to 0.4 N NaOH, then denatured

by boiling for 10 min and immediately chilled on ice. About

100 ml of the prepared amplicons was dot-blotted onto a nylon

membrane and aspirated through the membrane under a

vacuum. The wells were then rinsed twice with 300 ml 2� SSC,

aspirated through the manifold under a vacuum, and briefly air-

dried. The membrane was UV cross-linked and probed with

[a�32P] cDNA. The cDNA was prepared from 3 mg of total RNA

from oil palm tissues using the First Strand cDNA Synthesis Kit

(Invitrogen), and then radiolabeled with [a�32P] according to the

MegaprimeTM DNA Labeling System manual (Amersham Life

Science). Prehybridization and hybridization were carried out

using standard techniques [22]. Blots were exposed to Kodak

XAR-5 film for 12 hours to 3 days.

Northern analysesAbout 15 mg of total RNA from various oil palm tissues were

denatured in RNA loading buffer (48% formamide, 6.4% formal-

dehyde, 1� MOPS buffer, 5.3% glycerol and 0.02% Bromophenol

blue). The mixture was denatured by heating for 10 min at 658Cfollowed by immediate cooling. Denatured total RNA was sepa-

rated on a 1% formaldehyde gel using 1� MOPS Buffer (20 mM

Morpholinopropanesulfonic acid, 5 mM sodium acetate and 1 mM

Na2EDTA pH 7.0) as the electrophoresis buffer. Transfer of RNA to

a nylon membrane (Hybond N+ Amersham) was carried out using

the capillary transfer method. The membrane was UV cross-linked

and probed with [a�32P] DNA probe. The DNA was radiolabeled

with [a�32P] according to the MegaprimeTM DNA Labeling System

manual (Amersham Life Science). Prehybridization and hybridiza-

tion were carried out using standard techniques [22]. Blots were

exposed to Kodak XAR-5 film for a week.

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Genome walkingFour blunt-end digestions were performed using Dra1, EcoRV, PvuII

and Stu1. The digested DNA was purified and ligated to Genome

Walker Adaptors. About 100 ng of each DNA genomic library was

used as the DNA template for primary PCR reaction. PCR ampli-

fication was performed in reaction mixture containing 1� Advan-

tage 2 PCR reaction buffer, 0.2 M of each dNTP, 200 nM of each

primers, and 0.5–1 unit Advantage 2 Polymerase Mix. The ampli-

fication was performed in 7 cycles of 948C; 25 s, 728C; 3 min, and

followed by 32 cycles of 948C; 25 s, 678C; 3 min and 678C; 7 min.

The PCR products for each library were diluted 50 times and used

as the templates for secondary PCR. The secondary PCR amplifica-

tion was performed in 5 cycles of 948C; 25 s, 728C; 3 min, and

followed by 20 cycles of 948C; 25 s, 678C; 3 min and 678C; 7 min.

Cloning the DNA fragmentPurified PCR product was ligated into PCRII-topo vector (TOPO TA

Cloning Kit, Invitrogen Life Technologies) for further manipula-

tion. Ligation reactions consisted of 3 ml (about 30 ng) of purified

PCR product, 1 ml of salt solution (1.2 M NaCl, 0.06 M MgCl2), and

1 ml (10 ng) of vector plasmid. Sterile water was added to a final

volume of 6 ml. The mixture was incubated at room temperature

for 5–10 min and then mobilized into One Shot1 Chemically

Competent E. coli, according to the manufacturer’s protocol.

DNA sequencing for clone verificationPlasmid DNA was prepared using the Plasmid Mini Preparation Kit

(QIAGEN), according to the manufacturer’s protocol. Representa-

tive clones were sequenced using an automated DNA sequencer

(ABI PRISM Model 377 Version 3.4), and DNA sequences were

analyzed using VectorNTI software (Invitrogen). Following the

removal of unreadable and vector sequences, the analysis was

carried out to examine sequence alignment, ORF identification

and contig analysis and assembly. DNA and protein homology

searches against GenBank databases were performed using BLAST

2.0 [23]. Prediction of putative location of transcription start sites

was carried out using the Softberry database. Identification of cis-

acting regulatory elements was performed using MOTIF search at

publicly accessible databases. The databases used were Softberry

(http://www.softberry.com/berry.phtml), PLACE (http://www.

dna.affr.go.jp/PLACE) and PLANTCARE (http://bioinformatics.

psb.ugent.be/webtools/plantcare/html/).

Construction of transformation vectorThe uep1 coding region, starting from the translation start site, was

removed from the fragment to avoid undesired translation initia-

tion from the uep1 gene, as well as to ensure that gusA expression is

initiated using its own translational start codon. The promoter and

its 50 untranslated region were amplified from the plasmid

pGWUEP1. The amplification also introduced a Sph1 site at the

50 end and an Xba1 site at the 30 end. Purified PCR products were

then ligated into PCRII-topo vector (TOPO TA Cloning Kit, Invi-

trogen Life Technologies) to form pGWUEP2. pBI221 and uep1

promoter fragment (in pGWUEP2) were first digested with Sph1

and Xba1. Digestions were carried out in 100 ml reaction mixtures

containing 20 ml DNA, 1� buffer and 5 ml (10 units) of each

restriction enzyme and incubated overnight at 378C. The mixtures

were analyzed by agarose gel electrophoresis, and the fragments

New Biotechnology �Volume 27, Number 4 � September 2010 RESEARCH PAPER

were purified using the QIAquick Gel Extraction Kit (QIAGEN).

The ligation reaction was carried out using 1 ml (10 ng) of purified

pBI221 vector plasmid, 1� T4 ligation buffer, 1 unit of T4 ligase

and 5 ml (50 ng) of purified DNA insert. Sterile water was added to a

final volume of 20 ml, and the mixture was incubated overnight at

168C. The mixture was then transformed into One Shot1 Chemi-

cally Competent E. coli competent cells, according to the manu-

facturer’s protocol, and screened using restriction analysis. The

resulted clone was designated as pUEP1. The construction of

pUEP1 using pGWUEP2 is illustrated in Fig. 1.

FIGURE 1

Diagrammatical representation of the construction of pUEP1. The pGWUEP1 was d

replacing the CaMV35S to generate pUEP1. The arrows indicate the orientation o

Preparation of target materials for transformationOil palm tissues such as embryogenic calli, embryoid, immature

embryo, young leaflet from mature palm, green leaves, stem,

mesocarp nine weeks after anthesis (WAA) and tobacco green

leaves were cultured on agar solidified medium containing Mur-

ashige and Skoog (MS) macro- and micronutrient supplemented

with 1 mg/l napthaleneacetic acid (NAA) and 30 g/l sucrose. Meso-

carp tissues were sterilized in 20% bleach for 20 min and rinsed 3

times with sterile distilled water before being cultured. All explants

except for embryoid were cut into 5 mm � 5 mm disks before

igested with Sph1 and Xba1 and uep1 promoter was cloned into pBI221 by

f each DNA fragment assembled.

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being placed onto MS medium. All tissues were incubated in the

dark for 24 hours at 288C before bombardment.

Bombardment of oil palm tissuesParticle bombardment was conducted using the Bio-Rad PDS-1000

Hebiolistic particledelivery system (Bio-Rad,Hercules,CA,USA).To

each aliquot of 100 ml of gold particles, 20 mg of DNA, 100 ml of 2.5 M

CaCl2, and 40 ml of 0.1 M spermidine were added sequentially, with

continuous vortexing.Vortexing was continued for 3 min, followed

by centrifugation at 10,000 rpm for 10 s. The supernatant was

removed and the particles were washed twice with 500 ml of

100% ethanol, followed by centrifugation at 10,000 rpm for 60 s.

Finally, DNA-coated gold particles were resuspended in 120 ml of

absolute ethanol. For each bombardment, 6 ml of DNA-coated gold

particles was dispensed onto the center of a macrocarrier and dried

under sterile conditions. Target tissues were placed in the center of a

petri dish containing agar. Transformation was carried out using the

following parameters: bombardment pressure at 1100 psi; macro-

carrier to stopping screen distance at 6 mm; target plate distance to

stopping screen at 6 cm; chamber vacuum at 26 mmHg [20]. For oil

palm green leaf and mesocarp, the tissues were bombarded at 1350

and 1550 psi and 4.5 and 7.5 cmdistancesbetween stoppingplate to

target tissues, respectively. Other parameters used were as optimized

by Parveez (G.K.A. Parveez, Optimization of parameters involved in

transformation of oil palm using the biolistic method, PhD thesis,

Universiti Putra Malaysia, 1998). The bombarded tissues were then

incubated in the dark for 48 hours at 288C before GUShistochemical

analysis.

FIGURE 2

Photographic representations showing a reverse northern analysis to screen the ex

The membranes were hybridized with first strand cDNA from (a) mesocarp five wmesocarp 15WAA, (e) mesocarp 17 WAA, (f) kernel 14 WAA, (g) kernel 17WAA, (h)cDNA clone and ribosomal DNA, respectively.

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For the experiments to evaluate the effect of auxin and ABA

phytohormones on uep1 promoter activity, auxin treatment was

carried out by culturing the bombarded oil palm embryoid onto

MS media supplemented with 5 mg/l NAA for 48 hours. For ABA

response, the bombarded oil palm embryoids were placed on MS

medium supplemented with different contents of ABA (0, 4 and

8 mg/l) for two days. The analysis was conducted with four repli-

cates to increase its accuracy. The data were statistically analyzed

using Duncan Multiple Range Test (DMRT).

GUS histochemical assayGUS assay buffer (0.1 M NaPO4 buffer pH 7.0, 0.5 mM K-ferricya-

nide, 0.5 mM K-ferrocyanide, 0.01 M EDTA, 1 mg/ml X-gluc (5-

Bromo-4-Chloro-3-Indolyl-b-D-glucuronide), 1 ml/ml Triton-X

and 20% methanol (v/v)) [24] was filter-sterilized and stored at

�208C in the dark. Two days after bombardment, tissues were

stained overnight (16 hours) at 378C with GUS buffer. For green

tissues, samples were subsequently soaked in 70% ethanol and

incubated at 378C for one hour. This procedure was repeated 5

times or until the plant tissues became light green or clear. The

removal of chlorophyll improved the scoring of the blue staining

of the plant tissues. Blue spots were scored optically using a Nikon

UFX-DX microscope system.

Results and discussionIdentification of a constitutively expressing gene from oil palmReverse northern analysis was used to examine the expression

pattern of 73 EST clones that were generated through a microarray

pression pattern of 73 cDNA clones analyzed through a microarray approach.

eeks after anthesis (WAA), (b) mesocarp 9 WAA, (c) mesocarp 14 WAA, (d)frond, (i) flower. Blue and red arrows indicate the location of the pOPSFB-1301

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FIGURE 4

Photographic representation of a northern blot analysis for pOP-SFB1301

cDNA (a). Each lane contained 15 mg of total RNA prepared from differenttissues of oil palm. Lanes 1–4: mesocarp at 5, 9, 15, 19 WAA, lanes 5–7: kernel

at 12, 14, 17 WAA, lane 8: stem (shoot tips), lane 9: young leaf, lane 10: flower,

lane 11: root (from plantlet), lane 12: green leaf, lane 13: embryoid. Equal

loading of RNA was verified with 28S ribosomal DNA (b). Arrow indicates thesize of transcript.

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approach (Fig. 2). The EST clones used in this study were provided

by the Genomic Group of Malaysian Palm Oil Board (MPOB).

These clones were shown to be expressed in all tissues tested,

including inflorescence f18 (18 weeks after anthesis), embryoid

and callus. This observation was a good indicator that these EST

clones could encode constitutive genes in oil palm. However,

further analyses were needed to confirm that they were expressed

in other tissues.

In this study, the cDNA clone pOP-SFB1301, which encodes the

ubiquitin extension protein gene uep1, was strongly expressed in

all tissues tested. The analysis was carried out by normalizing the

intensities of cDNA expression to the intensity of ribosomal DNA.

The lowest value of cDNA expression obtained from the normal-

ization in any specific tissue was set as 1-fold. The result indicated

that the uep1 gene was expressed in all tissues tested with a range of

1.0–1.8-fold. The highest expression was detected in mesocarp at

week 5 (Fig. 3). Detailed sequence analysis showed that uep1

encodes a polyprotein consisting of 76 amino acid residues of

ubiquitin fused to a C-terminal extension. The extension was

predicted to be 80 residues and identified as a small subunit

ribosomal protein. Comparison of the pOP-SFB1301 cDNA

sequence to entries in GenBank revealed that this gene had

homologs in Arabidopsis, potato, barley, tomato, human and

rat, and that the domains are highly conserved among plant

and nonplant species.

In general, a ribosomal protein is any protein that conjugates

with ribosomal RNA (rRNA) to make up the ribosomal subunits.

In eukaryotes, the 40S subunit consists of 33 ribosomal proteins

and an 18S rRNA. The assembly of rRNAs and ribosomal proteins

to form 40S occurs within the nucleolus, a region of the nucleus

specialized for this purpose [25]. Ubiquitin extension protein

acts as ‘molecular chaperone’ that helps incorporate ribosomal

proteins into the nascent ribosome [26]. In yeast, deleting the

ubiquitin coding region from the ubiquitin extension protein

gene resulted in phenotype deficiencies such as slow growth,

abnormal RNA processing, and correspondingly low levels of 40S

ribosomal subunits [26]. Although it is clear that uep1 is coding

for a ribosomal protein involved in ribosome assembly, the

precise function and mechanism of uep1 action are still

unknown.

FIGURE 3

Graphical representation of the expression of pOPSFB-1301 cDNA in various

tissues of oil palm through reverse northern analysis. M5: mesocarp 5 WAA,M9: mesocarp 9 WAA, M14: mesocarp 14 WAA, M15: mesocarp 15 WAA, M17:

mesocarp 17 WAA, K14: kernel 14 WAA, K17: kernel 17 WAA, FR: frond and FL:

flower. A strong signal (relatively) was observed in all tissues tested.

Northern analyses were utilized to study the transcript size and

regulation of oil palm uep1. A transcript with a size of about 0.8 kb

was detected in total RNA hybridization of various oil palm tissues

including mesocarps, kernels, frond, young leaf, embryoids, root,

flower and stem. The RNA transcript was found to be most abun-

dant in young leaf, flower, root, stem and embryoids. High level of

transcript was also detected in early stages of mesocarp develop-

ment at 5–9 WAA. The level was slightly decreased at week 15–19

(Fig. 4). This slight variation could be caused by mRNA accumula-

tion in the different stages of tissue development. A previous study

showed that the gene is highly expressed in young tissues or tissues

containing rapidly dividing cells than in more mature tissues [21].

This accumulation is required for active protein synthesis during

the early stages of plant development. This pattern of expression

has also been observed in tomato [18], barley [19], potato [20] and

Arabidopsis [21]. However, uep1 expression in green mature leaves

was slightly lower than that observed in young leaves. In potato,

although the expression of GUS driven by an ubiquitin extension

protein promoter was relatively low in mature leaves, the tran-

scripts were increased in senescence leaves. This suggested that

UEP may be involved in the synthesis of proteins required for

senescence, or alternatively, that UEP may be required to supply

sufficient ubiquitin for protein degradation [20,27]. Surprisingly,

in kernels, both northern and reverse northern analyses revealed a

low level of uep1 transcript in all stages tested. The low expression

level of uep1 could be due to the lack of cellular division in kernels.

However, further analyses must be carried out to investigate the

authentic role of this gene in kernel development.

Although the uep1 levels varied slightly between different tis-

sues, this molecular analysis verified the constitutive status of the

uep1 gene. The results obtained from northern analyses also con-

curred with the reverse northern analyses. These results also

suggested that expression of this gene is very important through-

out the plant life cycle, coinciding with its role in ribosomal

biogenesis.

Isolation of a constitutive promoter from oil palmGenome walking procedure was employed to amplify the uep1

promoter region. A PCR product of about 1.1 kb, which included

part of the uep1 coding region, was obtained. The DNA sequence of

the fragment was analyzed for the identification of the promoter

region. Based on the analyses (NCBI, Softberry, PlantCare, PLACE

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FIGURE 5

DNA sequence and map of the oil palm uep1 genomic clone. The sequence is numbered from the 50 PvuII site. The UEP1 coding region begins at residue 829 and

extends to residue 1296. The predicted protein sequence is shown extending to the first stop codon 50 to the initiating codon, methionine (M). Position of putativetranscription start site (A) is indicated with large and bold font. The putative TATA box, CAAT box and other putative cis-elements are underlined and labeled.

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databases and VNTI software), the 828 bp sequence upstream of

the translational start site was identified as part of uep1 promoter.

The region includes the 50 untranslated region of the gene. The

putative transcription start site was predicted 100 nucleotides

upstream of the translational initiation site (Fig. 5). A potential

TATA box sequence was identified 30 bp upstream of the tran-

scription start site. Additionally computer analysis was used to

identify other features in the gene architecture that could con-

tribute to uep1 expression (Fig. 5). One of the important motifs

identified was a sequence that closely matched the consensus

sequence of upstream activation sites (UAS) in yeast ribosomal

protein genes. This sequence function is to promote transcription

[26] and was reported to be present in uep1 counterparts from other

plants including barley, maize and tomato. However, such UAS

sites have also been found within a few genes encoding nonribo-

somal protein [28]. The uep1 promoter region also contains T-rich

stretches that represent a second sequence motif characteristic of

ribosomal protein gene promoters [26] (Fig. 4). T-rich motifs

contribute to the high transcriptional yields of various ribosomal

protein gene promoters [29,30]. These TC-rich sequences were also

found in yeast [16] and barley [19] ubiquitin extension protein

promoters.

The putative promoter sequences were also analyzed using plant

databases to find other important cis-acting regulatory elements.

Examination of the nucleotide sequences upstream of the uep1

gene revealed that it contains multiple motifs as shown in Fig. 5. It

was observed that oil palm uep1 promoter contains sequences

associated with light-responsive elements (LRE) including GATA

box and GT-1 like elements. The LRE motifs are highly conserved

in photoregulated and generally required for high level, light-

regulated and tissue-specific expression [31].

In addition, uep1 promoter also contains other interesting

motifs such as ethylene- (ERE), abcisic acid- (ABRE), auxin-

(AuXRE) and water stress-responsive elements (MYB). This indi-

cated that UEP has a pivotal role in multiple hormonal signaling

pathways and can be activated by both abiotic and some physical

stresses. Garbarino and Belknap [20] reported that the ubiquitin

extension protein gene in potato tubers was strongly induced by

wounding [20]. The production of stress ethylene in wounded

tissues resulted in a large increase in metabolic activity [32], which

has been known to stimulate the biosynthesis of new ribosomal

components [20,33]. This promoter also contains other elements

that confer tissue- and cell-specific expression. Sequence analysis

does demonstrate the presence of motifs similar to root-specific

element, guard cell-specific expression and pollen-specific cis-act-

ing elements. Another interesting element is AT-1 motif, which

has also been found in barley, tomato and maize ubiquitin exten-

TABLE 1

Comparison of promoter strength on transient gusA gene expression

Promoter/construct Mean (standard error) of GUS foci

YMLP EC EM

Ubi1/pAHC25 9166.6 � 119.2 121.67 � 17.68 568.8 � 129.50

CaMV35S/pBI221 3504.2 � 133.3 100.33 � 52.1 314.0 � 56.50

uep1/pUEP1 1812.0 � 75.7 42.25 � 5.82 237.8 � 61.95

YMLP, young leaflet from mature palm; EC, embryogenic calli; EM, embryoid; ST, shoot tip (m

sion protein promoters, as well as photoregulated genes, nodulin-

encoding genes and seed-protein encoding genes [17].

In conclusion, the data presented clearly indicate that the oil

palm uep1 promoter is controlled by multiple cis-acting regulatory

elements or motifs that confer constitutive expression. Studies on

the regulation of the nominally constitutive CaMV35S promoter

in transgenic plants have shown that constitutive promoters

might contain multiple cis-acting elements, each of which inter-

acts individually with cell- or tissue-specific trans-acting factors.

The combined activities of individual cis-acting elements confer

constitutive gene expression in most tissues [34]. Therefore, it is

possible that the presence of multiple regulatory elements may

enable uep1 expression to be maintained under various light

influences, or if the abundance of specific trans-acting factors

varies during development. Moreover, such a reiteration of genetic

information may enable the gene to compensate for changing

environmental conditions and developmental cues [35].

Evaluation of promoter activityThe activity of oil palm uep1 promoter was evaluated through

transient expression study by bombarding the pUEP1 vector into

different oil palm target tissues. For comparison, the oil palm

tissues were also bombarded with pAHC25 that carries gusA and

bar genes driven by maize polyubiquitin promoter and the original

pBI221 plasmid DNA that carries the gusA gene driven by

CaMV35S promoter. pAHC25 was used as the positive control

as maize polyubiquitin promoter has been extensively and suc-

cessfully used to express chimeric genes in monocot transforma-

tion studies [36]. More importantly, the promoter is capable of

driving high expression of GUS reporter gene in oil palm tissues

[37]. By contrast, CaMV35S promoter was also used as positive

control as it is capable of driving expression of transgenes in

monocot and dicot plant systems [38]. The constructs were bom-

barded in oil palm tissues and tobacco green leaf. These tissues

were also bombarded with gold particles without DNA as an

additional control. The GUS expression was detected in all tissues

tested as indicated by the presence of blue spots. No GUS expres-

sion was detected in oil palm tissues bombarded with gold parti-

cles. This observation clearly indicated that the blue spots

observed were due to introduced genes. GUS activity was deter-

mined by counting the GUS-positive spots optically. Each blue

spot detected, whether in a single cell or a group of cell was

considered as one expression unit as defined by Klein et al. [39].

The data collected were then summarized in the form of mean

comparison and standard deviation as shown in Table 1. Histo-

chemical GUS assay indicated that uep1 promoter was capable of

driving GUS expression in all tissues tested including young leaflet

and GUS activity in oil palm tissues two days after bombardment

GL ST MS TB IE

132 � 12.02 62.0 � 27.7 105.5 � 9.54 15 � 11.0 148 � 28.1

81.4 � 7.07 85.7 � 3.23 70.2�5.61 33 � 9.71 124 � 22.7

54.5 � 9.54 37.0 � 11.2 43.6 � 18.22 12.5 � 2.5 97 � 16.3

eristematic tissues); MS, mesocarp; TB, tobacco; IE, embryogenic calli.

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FIGURE 6

Photographic representations of the comparison of transient histochemical assay in various oil palm tissues and tobacco bombarded with plasmid carrying gusA

gene driven by different promoters. (a) None (bombarded without plasmid DNA), (b) pAHC25 (Ubi1), (c) pBI221 (CaMV35S) and (d) pUEP1 (uep1).

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FIGURE 7

GUS activity induced by (a) auxin and (b) abscisic acid in oil palm embryoid

bombarded with pUEP1 construct. Means with the same letter are not

significantly different at P < 0.05 according to Duncan’s Multiple Range Test.

Bars represent standard error.

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from mature palm, embryoid, green leaves (from plantlet), stem

(from plantlet) and mesocarp (Fig. 6). The uep1 promoter was also

capable of driving gusA expression in all cross-section (vertically)

part of immature embryo including the area containing meriste-

mic tissue.

Overall, these experiments indicated that in oil palm, except for

shoot tip, the highest GUS expression was obtained in tissues

bombarded with constructs driven by maize ubi1, followed by

the CaMV35S and uep1 promoters. This result concurred with

Chowdhury et al. [37], who reported that the activity of maize

ubi1 was superior to CaMV35S in all oil palm immature embryo,

young leaf and embrogenic calli. Moreover, Callis et al. [21] found

that expression of Arabidopsis ubiquitin extension protein (UBQ1

and UBQ6) promoters were also slightly lower than that of

CaMV35S.

The activity of oil palm uep1 promoter was also examined in a

dicot system by bombarding this promoter construct into

tobacco green leaves tissue (Fig. 6). As expected, high GUS

expression was observed in tissues bombarded with CaMV35S

promoter. This could be because the CaMV35S promoter is more

effective in dicots than monocots. Interestingly, the uep1 pro-

moter could drive GUS expression in tobacco indicating that this

promoter could also be used in dicot system. The activity of oil

palm uep1 was similar to ubi1 promoter in terms of the number of

blue spots.

Generally, results showed that the strength of oil palm uep1

promoter is slightly lower than the other promoters used, parti-

cularly to pAHC25. The differences in the promoter activities

could be due to the presence of an intron region located adjacent

to the maize ubi1 promoter. Numerous studies have shown that

the high expression capacity of constitutive promoters in mono-

cots is usually caused by the presence of an intron located in the 50

untranslated region [40]. It has been suggested that the 50 intron

may be required in in vivo for efficient mRNA splicing [41]. How-

ever, uep1 does not contain a native intron for this purpose.

Therefore, it could be suggested that the relatively low strength

of the uep1 promoter may be due to the lack of intron in the uep1

gene, particularly in the pUEP1 transformation vector. Sivamani

and Qu [42] reported that when the rice polyubiquitin intron was

placed behind the rice Act1 promoter (without its own 50 UTR), the

promoter activity was enhanced by 8–9-fold. Thus, the efficiency

of the uep1 promoter could potentially be increased by the inser-

tion of an intron into the construct.

The activity of the uep1 promoter could also be significantly

enhanced if the gusA coding sequence was fused in-frame to its

ubiquitin monomer coding sequence. This strategy slightly

increases the activity of ubiquitin promoters in both dicots

and monocots. It has been reported that, when fused to a 76-

amino-acid ubiquitin monomer sequence, potato uep-driven

GUS expression was 5–10 times higher than constructs that

did not contain fused ubiquitin monomer [20]. Moreover, the

activity of a rice polyubiquitin promoter was significantly

enhanced when the reporter gene was fused to both its 50UTR

and a nine-amino-acid coding region of ubiquitin monomer

[42]. The inclusion of the ubiquitin monomer coding region is

thought to increase either transcription of the reporter gene or

message stability [20]. The ubiquitin moiety is subsequently

removed by ubiquitin-C-terminal hydrolases or de-ubiquitinat-

ing enzymes, the specific proteases that release the ubiquitin

monomers, so that only the native reporter products accumulate

in vivo [43,44].

Activity of the uep1 promoter in response to exogenous auxinand abcisic acidAuxin and ABA phytohormones play a pivotal role in many

physiological and developmental processes in plants. As uep1

consists of cis-acting elements associated with auxin (AuXRE)

located at �580 and abscisic acid (�ABRE) at �604 upstream of

the promoter region, further study was carried out to investigate

whether or not the promoter transient activity could be elevated

by the hormone induction. An increased level of gusA expression

was observed in both treatments, indicating an induction of

promoter activity by the hormones (Fig. 7). However, the incre-

ment of gusA activity by hormone treatments was not significantly

different (Fig. 6). This could be because the TGTCTC auxin

response (AuxRE) and ABRE responsive elements were only present

in a single copy in oil palm uep1 promoter region. Therefore, it is

possibly not sufficient for increasing auxin- or ABA-mediated

induction of gus transcription. The GH3 auxin-regulated genes

were found to consist of at least three auxin response elements

(AuxREs) which function independently to one another to confer

auxin inducibility [45]. Studies have also indicated that to stimu-

late ABA responsiveness, a minimal ABA-responsive complex

(ABRC) which contains multiple ABREs or the combination of

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an ABRE with coupling element (CE) was necessary in minimal

promoter region [46,47]. By contrast, the nonsignificant difference

in transient activity after hormone treatment may also be due to

short exposure duration. Testing the hormone effects on stably

transformed model plants, such as Arabidopsis or tobacco may be

more practical and useful.

ConclusionIn this study, uep1 promoter was capable of directing the expres-

sion of a readily detectable level of GUS in all tissues tested. The

result demonstrated its role as a constitutive promoter in oil palm.

This study has shown that the endogenous uep1 promoter could be

used as a crucial biotechnology tool for producing transgenic oil

palm, whereby constitutive and high level expression of trans-

genes may be required. Although the strength of the promoter was

298 www.elsevier.com/locate/nbt

relatively lower than positive controls, modification of uep1 pro-

moter by the addition of intron and ubiquitin monomer could

potentially increase the strength of the oil palm uep1 promoter.

AcknowledgementsThe authors wish to thank the Director-General of MPOB for

permission to publish this paper. We wish to thank Dr Leslie Low

Eng Ti and members of Genomic Group, MPOB for their kind

consent in providing EST clones of oil palm. We owe special thanks

to Breeding Group for providing oil palm tissues and also to Dr

Noor Azmi Shaharudin, Gene Expression Group, MPOB. Finally

the authors would like to acknowledge Dr Omar Abdul Rasid and

all staff of Genetic Transformation Laboratory and School of

Biosciences and Biotechnology, Faculty of Science and

Technology, UKM for their help and assistance.

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