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Hindawi Publishing Corporation Sequencing Volume 2013, Article ID 756983, 10 pages http://dx.doi.org/10.1155/2013/756983 Research Article Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein from Plantago ovata Forsk Amitava Moulick, 1 Debashis Mukhopadhyay, 2 Shonima Talapatra, 1 Nirmalya Ghoshal, 1 and Sarmistha Sen Raychaudhuri 1 1 Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, 92, APC Road, Kolkata 700009, India 2 Structural Genomics Section, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India Correspondence should be addressed to Sarmistha Sen Raychaudhuri; sarmistha rc@rediffmail.com Received 28 September 2012; Revised 31 January 2013; Accepted 11 February 2013 Academic Editor: Alfredo Ciccodicola Copyright © 2013 Amitava Moulick et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Plantago ovata Forsk is a medicinally important plant. Metallothioneins are cysteine rich proteins involved in the detoxification of heavy metals. Molecular cloning and modeling of MT from P. ovata is not reported yet. e present investigation will describe the isolation, structure prediction, characterization, and expression under copper stress of type 2 metallothionein (MT2) from this species. e gene of the protein comprises three exons and two introns. e deduced protein sequence contains 81 amino acids with a calculated molecular weight of about 8.1 kDa and a theoretical pI value of 4.77. e transcript level of this protein was increased in response to copper stress. Homology modeling was used to construct a three-dimensional structure of P. ovata MT2. e 3D structure model of P. ovata MT2 will provide a significant clue for further structural and functional study of this protein. 1. Introduction e seed husk of Plantago ovata Forsk has been used as a dietary fiber for a long time, and more recently it has been shown to reduce the development of endothelial dysfunction, hypertension, and obesity [1]. e plant has been shown also to reduce carbohydrate absorption and postprandial rise of glucose and insulin levels in type 2 diabetes patients [2]. It also lowers plasma lipids by altering hepatic and bile acid metabolism [3]. Studies on the effect of aqueous extracts of P. ovata in patients with diabetes found that it can reduce hyperglycemia via inhibition of intestinal glucose absorption and enhancement of motility [4]. Heavy metal ions play essential roles in many physio- logical processes. Among these, copper (Cu) is an essential micronutrient in plants. It is a component of several electron transport enzymes and is involved in catalyzing the redox reactions in mitochondria and chloroplasts [5]. Copper also plays important roles in respiration, carbohydrate distri- bution, protein metabolism, water relations, reproduction, and disease resistance [6]. However, at high concentration, this metal can inhibit plant growth, causes degradation of chlorophyll, and impedes photosystem (PS) II activity [7, 8]. Soil can be naturally rich in heavy metals [6] which become the major pollutant by industrial processes, such as steel production and petroleum processing [9]. Copper is considered to be one of the most important pollutants of agricultural soils [6]. Sources of Cu contamination include mining and smelting; urban, industrial, and agricultural wastes; and the use of fungicides and herbicides [10]. A general mechanism of copper toxicity may be the generation of harmful reactive oxygen species (ROSs) which can damage biological molecules (DNA, RNA, and proteins) and mem- branes by inducing lipid peroxidation [5]. In response to toxic levels of heavy metals, plants evolved a suitable mechanism that controls the uptake and accumulation of both essential and nonessential heavy metals by synthesizing cysteine-rich, metal-binding peptides like metallothioneins which in turn helps in detoxification of heavy metals by chelation and sequestration in the vacuole [11, 12]. Metallothioneins (MTs) can protect cells against the toxic effects of copper by chelating heavy metal [13]. MTs are
Transcript
Page 1: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

Hindawi Publishing CorporationSequencingVolume 2013 Article ID 756983 10 pageshttpdxdoiorg1011552013756983

Research ArticleMolecular Cloning Modeling and Characterization ofType 2 Metallothionein from Plantago ovata Forsk

Amitava Moulick1 Debashis Mukhopadhyay2 Shonima Talapatra1

Nirmalya Ghoshal1 and Sarmistha Sen Raychaudhuri1

1 Department of Biophysics Molecular Biology and Bioinformatics University of Calcutta 92 APC Road Kolkata 700009 India2 Structural Genomics Section Saha Institute of Nuclear Physics 1AF Bidhan Nagar Kolkata 700 064 India

Correspondence should be addressed to Sarmistha Sen Raychaudhuri sarmistha rcrediffmailcom

Received 28 September 2012 Revised 31 January 2013 Accepted 11 February 2013

Academic Editor Alfredo Ciccodicola

Copyright copy 2013 Amitava Moulick et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Plantago ovata Forsk is a medicinally important plant Metallothioneins are cysteine rich proteins involved in the detoxificationof heavy metals Molecular cloning and modeling of MT from P ovata is not reported yet The present investigation will describethe isolation structure prediction characterization and expression under copper stress of type 2 metallothionein (MT2) from thisspeciesThe gene of the protein comprises three exons and two intronsThe deduced protein sequence contains 81 amino acids witha calculated molecular weight of about 81 kDa and a theoretical pI value of 477 The transcript level of this protein was increasedin response to copper stress Homology modeling was used to construct a three-dimensional structure of P ovata MT2 The 3Dstructure model of P ovataMT2 will provide a significant clue for further structural and functional study of this protein

1 Introduction

The seed husk of Plantago ovata Forsk has been used as adietary fiber for a long time and more recently it has beenshown to reduce the development of endothelial dysfunctionhypertension and obesity [1] The plant has been shown alsoto reduce carbohydrate absorption and postprandial rise ofglucose and insulin levels in type 2 diabetes patients [2] Italso lowers plasma lipids by altering hepatic and bile acidmetabolism [3] Studies on the effect of aqueous extracts ofP ovata in patients with diabetes found that it can reducehyperglycemia via inhibition of intestinal glucose absorptionand enhancement of motility [4]

Heavy metal ions play essential roles in many physio-logical processes Among these copper (Cu) is an essentialmicronutrient in plants It is a component of several electrontransport enzymes and is involved in catalyzing the redoxreactions in mitochondria and chloroplasts [5] Copper alsoplays important roles in respiration carbohydrate distri-bution protein metabolism water relations reproductionand disease resistance [6] However at high concentration

this metal can inhibit plant growth causes degradation ofchlorophyll and impedes photosystem (PS) II activity [78] Soil can be naturally rich in heavy metals [6] whichbecome the major pollutant by industrial processes such assteel production and petroleum processing [9] Copper isconsidered to be one of the most important pollutants ofagricultural soils [6] Sources of Cu contamination includemining and smelting urban industrial and agriculturalwastes and the use of fungicides and herbicides [10] Ageneral mechanism of copper toxicity may be the generationof harmful reactive oxygen species (ROSs) which can damagebiological molecules (DNA RNA and proteins) and mem-branes by inducing lipid peroxidation [5] In response to toxiclevels of heavy metals plants evolved a suitable mechanismthat controls the uptake and accumulation of both essentialand nonessential heavy metals by synthesizing cysteine-richmetal-binding peptides like metallothioneins which in turnhelps in detoxification of heavy metals by chelation andsequestration in the vacuole [11 12]

Metallothioneins (MTs) can protect cells against the toxiceffects of copper by chelating heavy metal [13] MTs are

2 Sequencing

proteins rich in cysteine (Cys or C) residues that bindheavy metals and play a role in buffering the intracellularconcentration of free thiophilic metal ions such as Cu Znand Cd [14] MTs are widely distributed among the animaland plant kingdom MTs are low molecular weight proteinswhich contain two metal-binding cysteine-rich domainslinked by a Cys-free spacer which varies between plant andanimal [12] MTs also play important roles in regulationof metalloenzymes and transcription factors scavenging ofreactive oxygen species metabolism of metallodrugs andalkylating agents response to stress conditions and apop-tosis [15] Classically MTs were grouped into three classesaccording to their sequence similarities [12] Afterward itwas classified into 15 families on the basis of taxonomicrelationships with plant MTs being placed into Family 15[16] All plant MTs (class II) were further distributed intofour types according to the distribution of cysteine residuesin the amino- and carboxy-terminal regions similarities Thespacer region separating these domains in Type 2 MTs ismuch more variable between species [12] In the presentstudy we found that amino acid sequence of Plantagoovata MT2 is very similar to Plantago major MT2 andthe only difference between them was in the spacer regionSeveral plant MT proteins are found in the data base butvery little experimental structural information has yet beenreported so far due to difficulties in protein purificationThe structure is very useful to understand the biologicalfeatures and functions of a protein Therefore theoreticalmethod is needed to predict the three-dimensional structureof proteins

A novel finding of metallothionein type 2 gene fromPlantago ovata is reported here The objective of the presentwork is to investigate the MT2 expression pattern againstcopper toxicity The isolated and molecular characterizedMT2 transcript and its predicted structure contribute toa better understanding of MT2 on the basis of structure-function relationship in an important medicinal plant like Povata

2 Materials and Method

21 Plant Material and Treatment Seeds of Plantago ovatawere collected from Gujarat India The seeds were sterilizedin 10 sodium hypochlorite and washed five times to washoff the excess bleach totally from the seed surface andimbibed in sterilized distilled water Next day the seedswere transferred to agar-sucrose media containing 3 (wv)sucrose (SRL Mumbai India) and 09 (wv) agar (SRLMumbai India) for germination according to the methodas described by Das and Sen Raychaudhuri [17] Eight-day-old seedlings were transferred to liquidMurashige and Skoog(MS)medium (Himedia Mumbai India) supplemented withdifferent concentrations ofCuCl

2(20120583M40 120583M and 80 120583M)

along with control The specified doses have been selectedafter considering the LD

50of copper in P ovataThe LD

50of

copper was found to be 120120583M in case of P ovate and hencea range of lower doses have been selected [18] CuCl

2(copper

(II) Chloride) (Merck Darmstadt Germany) treatment was

carried out for 24 h and 72 h to observe the changes of MTtranscript levels

22 Genomic DNA Extraction Genomic DNA was isolatedfrom seedlings following the standard protocol of Edwards etal [19] Seedlings were crushed gently using DNA extractionbuffer followed by a centrifugation to remove the tissuefragments Supernatant was taken and equal volume ofphenol chloroform (1 1) (SRL Mumbai India) was addedto it After that a centrifugation was carried out and thesupernatant was washed with equal volume of chloroformFinally the clear upper aqueous phase was collected and 3Mammonium acetate and equal volume of isopropanol (SRLMumbai India) were added and mixed DNA was spooledwith glass capillary and washed with 70 ethanol (SRLMumbai India) Subsequently the DNA was air dried anddissolved in sterile triple distilled water The DNA was run in1 agarose gel and spectrophotometrically scanned to checkthe quality and quantity

23 Total RNA Extraction Total RNA was extracted fromseedlings using RNeasy Plant Mini Kit (Qiagen HamburgGermany) according to the manufacturerrsquos protocol All theglass goods were treated with diethyl pyrocarbonate (SigmaSt Louis USA) prior to RNA extraction RNAwas quantifiedspectrophotometrically The extracted RNA was preserved atminus70∘C

24 Primer Designing The primers for P ovata MT2 weredesigned based on published sequences Published sequenceswere taken using Blast search and they were aligned usingClustalW Primers were designed from those regions ofPlantago major MT2 sequence showing best homology withother published sequences The forward and reverse primersof MT2 were of 23 and 22 bases respectively The primerswere designed in such away so that theirmelting temperaturewould be close The sequences of forward and reverse MT2primers were 51015840-TCTTGCTGCAACGGAAACTGTGG-31015840and 51015840-AGTTGTCACCGCACTTGCACCC-31015840 respectivelyUbiquitin was taken as a housekeeping gene [20 21] tonormalize the reverse transcription PCR analysis Thesequences of ubiquitin primers were designed in thesame procedure as described above The sequences offorward and reverse primer of P ovata ubiquitin were51015840-TGAAAACTTTTACAGGCAAGACC-31015840 and 51015840-GAC-GGAGTACCAAATGGAGAGTG-31015840 respectively

25 Polymerase Chain Reaction (PCR) and Reverse Transcrip-tion Polymerase Chain Reaction (RT-PCR) PCR and RT-PCR were carried out using designed primers Taq enzymeTaq buffer and MgCl

2(magnesium chloride) used in PCR

were brought from Genei (Bangalore India) and were usedaccording to themanufacturerrsquos protocolThe thermal profilefor PCR amplification of MT2 was as follows 94∘C for2min followed by 35 cycles at 94∘C for 1min 62∘C for30 sec and 72∘C for 1min 30 sec and a final extension at72∘C for 10min RT-PCR was carried out using One StepRT-PCR Kit (Qiagen Hamburg Germany) according to

Sequencing 3

the manufacturerrsquos protocol with the designed primers Thereverse transcription experiments were carried out usingequal amounts of total RNA (2 120583g) extracted from copperstressed and control seedlings Simultaneously RT-PCR wasalso carried out for ubiquitin in the same conditions of exper-iment This step was essential to estimate the efficiency ofreverse transcription system after 25 cycles of amplificationPCR and RT-PCR was carried out using GeneAmp PCRSystem (Applied Biosystems Carlsbad USA) The thermalprofile for RT-PCR amplification of MT2 was as follows 50∘Cfor 30min 95∘C for 15min followed by 25 cycles at 94∘Cfor 1min 62∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min The RT-PCR cycle forubiquitin was 50∘C for 30min 95∘C for 15min followedby 25 cycles at 94∘C for 1min 57∘C for 1min and 72∘Cfor 1min 30 sec and a final extension at 72∘C for 10minAliquots of 25 120583L of the PCR and RT-PCR products wererun on 15 and 2 agarose gel respectively The gels werestained with ethidium bromide and photographs were takenusing aGelDocumentation System (BioRadHercules USA)All RT-PCR reactions were replicated at least thrice fromthree independent RNA preparations The pixel intensityof the band in gel was quantified using ImageJ softwareThe obtained results were subjected to statistical analysisusing Kyplot and Microsoft Excel All data were expressed asmean plusmn standard error (SE) These data were analyzed by ananalysis of variance (ANOVA) Groupmeans were comparedusing Studentrsquos 119905 test if significance was found in ANOVADifferences of the data were considered significant when 119875 le005

26 Cloning and Sequence Analysis Cloning and SequenceAnalysis of The PCR product of metallothionein type 2 waspurified by PCR clean-up kit (Chromus Biotech BengaluruIndia) according to the manufacturerrsquos protocol TheMetallothionein gene fragment was cloned into the vectorpTZ57RT using the protocol provided in the InsTAclonePCR cloning Kit (Fermentas St Leon-Rot Germany) Ecoli XL 1- blue was used as a host strain After cloning thepurified plasmid it was sequenced using universal primersThe primer sequences are 51015840-GTAAAACGACGGCCAGT-31015840 and 51015840-CAGGAAACAGC- TATGAC-31015840 In case ofubiquitin the PCR product was sequenced using genespecific primers The sequences were analyzed usingseveral programs such as BLASTN BLASTX BLASTPand ClustalW [22 23] The deduced amino acid sequenceof P ovata MT2 was analyzed to detect the N-terminalsorting signal using iPSORT (httpipsorthgcjp) andto determine the pI value and molecular weight usingExPASy (httpwwwexpasychtools) [22] To determinethe hydrophobic nature of the amino acids in the protein ahydropathy plot was created using ExPASy-ProtScale (httpwebexpasyorgprotscale) choosing Kyte amp Doolittle scaledefault parameters and window size 9 The disulfidebond partners in the P ovata MT2 were predictedusing DiANNA server (httpclaviusbcedusimclotelabDiANNAmainhtml) For better estimation of therelationships between P ovata MT2 and other reported

MTs a phylogenetic tree was generated using Phylip369 package (httpevolutiongeneticswashingtoneduphyliphtml) enabling 100 bootstraps Unique metalloth-ionein sequences were selected from UniProtSwiss-Protknowledgebase (httpwwwuniprotorgdocsmetallotxt) [24 25] One sequence from each metallothioneinsubdivision was taken along with all plant MT (Family 15)to generate a significant phylogenetic tree As primers weredesigned from the available closest species Plantago majoramino acid sequences of metallothionein of this specieswere included in the phylogenetic tree Sequence file (Phylipformat) was run using seqboot choosing 100 replicate andall other default parameters The outfile of this programwas run using protpars for parsimony based phylogeneticanalysis keeping the number of jumbles set to 10 and all otherparameters as default the output tree of this programwas runusing consense Finally the unrooted tree was constructedusing njplot (httppbiluniv-lyon1frsoftwarenjplothtml)

27 Expression Analysis Using Real-Time PCR Formetalloth-ionein type 2 gene expression analysis first strand cDNAwassynthesized from total RNA (sim2120583g) of all the treated (for72 h with different concentrations of CuCl

2) samples using

high-capacity RNA-to-cDNA kit (Applied Biosystems USA)according to the manufacturerrsquos instruction Real-time PCRwas carried out in Step One Plus Real-Time PCR thermocy-cler (Applied Biosystems UK) Each reaction contained 2XPower Cyber Green PCR master mix (Applied BiosystemsUK) diluted cDNA and 10 pmol gene specific primers Thesequences of forward and reverse primers used in real-time PCR were 51015840-TCTTGCTGCAACGGAAACTGTGG-31015840and 51015840-AGTTGTCACCGCACTTGCACCC-31015840 respectivelyFollowing thermal conditions have been used heating at95∘C for 10min 40 cycles of denaturation at 95∘C for 15 secannealing and extension at 60∘C for 1min followed by amelt curve analysis to ensure the amplification of only thedesired amplicon A gene encoding P ovata actin was usedas endogenous control The primers used to amplify actinwere 51015840-ATCATGAAGTGTGATGTTGA-31015840 (forward) and51015840-ACCTTAATCTTCATGCTGCC-31015840 (reverse) The relativegene expression was conducted using the 2minusΔΔCT method[26] The data of relative gene expression were analyzed byStep One software version 21 (Applied Biosystems UK) Allthe experiments were performed in triplicate A negativecontrol was included in each reaction The results presentedhere are means of three replicates and the bars indicatestandard deviation Statistical significance of real-time exper-iment results were analyzed by analysis of variance (ANOVA)using Kyplot software

28 Molecular Modeling of P ovata MT2 The deducedamino acid sequence of P ovata MT2 (complete codingregion) was submitted to ModWeb server (httpsmod-basecompbioucsfeduscgimodwebcgi) for comparativeprotein structure modeling choosing default parameters[27] ModWeb performs automated comparative modelingwhich relies on PSI-BLAST IMPALA and MODELLER[28] This server uses ModPipe (version SVNr13401348M)

4 Sequencing

PoMT2 gene (partial CDS)

1

46

100159218

264

309368427486

536

(a)

PoMT2 mRNA (complete CDS)

1

46

91

136

181

226lowast

(b)

Figure 1 Nucleotide and deduced amino acid sequences of PoMT2 Two introns interrupt the coding region at the points marked by arrows(a) Partial coding region encodes a protein of 71 amino acids (GenBankAccession no GU596501 andGU596503) (b) Complete coding regionencodes a protein of 81 amino acids

1

45

90

135

180

P ovata ubiquitin mRNA (partial CDS)

Figure 2 Nucleotide and deduced amino acid sequences of P ovata ubiquitin (GenBank Accession no JQ419758) Partial coding regionencodes a protein of 67 amino acids

as underlying software pipeline Human ADAM22 chainA (Protein Data Bank ID 3G5C) was used as templatestructure (template region 447ndash523) for modeling of Povata MT2 The structure was reconstructed using theDeepViewSwiss-PdbViewer37 [15]

3 Result and Discussion

31 Cloning and Sequence Analysis PCR was carried outusing genomic DNA and a 578 bp fragment was found This

fragment was purified cloned and sequenced (Figure 1(a))On the other hand RT-PCR was carried out using totalRNA and a 214 bp fragment was found which was alsopurified cloned and sequenced (Figure 1(a)) Comparingboth the sequences it was found that the type 2 metal-lothionein gene of P ovata has 3 exons (0ndash63 222ndash308and 515ndash577) and 2 introns (64ndash221 and 309ndash514) in itssequence MT2 of some other plant species also show 3 exonsand 2 introns like Helianthus annuus (GenBank accessionno EF431954) Typha angustifolia (GenBank accession no

Sequencing 5

1 2 3 4 1 2 3 4

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

lowast lowast

lowastlowastlowast

lowastlowast

24 h 72 h

Pixe

l int

ensit

y (A

U)

Pixe

l int

ensit

y (A

U)

(a)

(b)

(c)

(d)

(e)

Figure 3 Results from reverse transcription PCR analysis Upper panel showing RT-PCR band in agarose gel of metallothionein type 2((a)(c)) and ubiquitin ((b)(d)) transcripts after treatment with different concentrations of CuCl

2(0 20 40 and 80 120583M in lane no 1 2 3

and 4 resp) Lower panel showing densitometry graph of corresponding band of MT2 Data represent the mean plusmn SE Asterisks indicatesignificant differences at 119875 lt 005 (lowast) 119875 lt 001 (lowastlowast) or 119875 lt 0001 (lowastlowastlowast) compared to respective controls AU means arbitrary unit

EF543085) and Hordeum vulgare (GenBank accession noJN997433)

After splicing out of the introns P ovata forms a 214 bpMT2 mRNA This sequence was searched for similar othersequences with the help of BLAST tool and many othersequences of metallothionein type 2 were found with 75ndash90 of sequence identity for example Plantago major MT2Avicennia marina MT2 and Ilex paraguariensis (GenBankaccession numbers are AJ843994 AF333385 and JX271039resp) The deduced amino acid sequence was also searchedby BLASTp tool to confirm that the obtained sequence wasof metallothionein type 2 The resultant nucleotide sequencewas aligned with some other reported metallothionein type 2with the help of ClustalW and similarity was found BLASTpanalysis of the deduced amino acid sequence also showedthat the fragment conformed to metallothionein type 2 superfamily Finally it was confirmed that the cloned sequence

was of P ovata metallothionein type 2 The successfullyisolated partial coding regions of MT2 gene and mRNAsequences of P ovata were submitted to GenBank with theaccession numbers GU596501 and GU596503 respectivelyand these were the first sequences of metallothionein fromthis species A later study by our group has sequenced thecomplete coding regions of MT2 from P ovata (PoMT2)(Figure 1(b)) (submitted to GenBank having accession noKC414846) encoding an 81-amino-acid protein with a cal-culated molecular weight of about 81 kDa and a theoreticalpI value of 477 The primer sequences used here were51015840-ATGTCTTGCTGCAACGGAAACT-31015840 (forward) and 51015840-CTATTTGCAATTGCATGGATTG-31015840 (reverse) The ther-mal profile for RT-PCR reaction was as follows 50∘C for30min 95∘C for 15min followed by 25 cycles at 94∘C for1min 54∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min Analysis of this PoMT2

6 Sequencing

6

5

4

3

2

1

0

Nor

mal

ized

relat

ive e

xpre

ssio

n

Control 20 120583M 40 120583M 80 120583MCuCl2 concentration in media

Figure 4 Real-time PCR expression profile of PoMT2 normalizedto the housekeeping gene actin under 0 20 40 and 80120583M copperstress The results indicate means of three replicates with theirstandard deviation

using iPSORT predicted that it does not have any mitochon-drial targeting peptide or chloroplast transit peptide UsingDiANNA server it was found that the PoMT2 has 7 predicteddisulfide bond between 3ndash8 4ndash75 10ndash67 14ndash20 16ndash78 23ndash80and 69ndash73 amino acid positions The terminal domain con-tained 0 aromatic 263 nonpolar aliphatic 605 polarand 105 charged amino acids In case of spacer region thesevalues were 93 419 302 and 186 respectively Thisprotein sequence of PoMT2 was compared using ClustalWand Blastp indicates that PoMT2 has 84 sequence identityto theMT2 encoded by Plantagomajor followed byAvicenniamarina 72 identity and Citrullus lanatus at 70 It hasbeen reported that class I MTs contain 20 highly conservedC residues showing homology to mammalian MTs whereasMTs without this strict arrangement of cysteines are referredto as class II MTs These MTs do not show homology tomammalian MTs Type 2 MTs contain CC CXC and CXXCmotifs in the N-terminal and CXC motif in the C-terminaldomain X can be any amino acid other than cysteine It alsocontains a CGGCmotif at the end of theN-terminal cysteine-rich domain and a spacer of approximately 40 amino acidresidues [12 15] In the present experiment PoMT2 followsall these criteria confirming that the isolated metallothioneinis a class II Type 2 plant metallothionein

The reverse transcription PCR product for ubiquitinwas sequenced A 204 bp sequence was obtained (Figure 2)(encoding 67 amino acids) which was searched for similarother sequences with the help of BLAST tool as describedabove It was found that the cloned sequence showed a goodsimilarity with some other published ubiquitin sequencesFor further confirmation the deduced amino acid sequencewas also searched by BLASTp tool and 99 sequenceidentity was found with other reported ubiquitin sequencesof Arabidopsis thaliana Plantago major Musa acuminateand Medicago truncatula (GenBank accession numbers areAAB95251 CAH56488 AAQ07454 and AET01627 resp)Finally it was submitted to GenBank with the accession

number JQ419758 It was the first ubiquitin sequence of Povata

32 Expression of PoMT2 Transcript in Different Stress Condi-tion To characterize PoMT2 transcript accumulation totalRNA extracted from various stress conditions were subjectedto reverse transcription (RT)-PCR reaction The seedlingswere treated with 0 20 40 and 80 120583M of CuCl

2solutions

for 24 h and 72 h (Figure 3) In each case 25 cycles of PCRreaction with first strand cDNA was carried out All thereactions showed the presence of a 214 bp bandThe intensityof the band was maximum in 40M CuCl

2treated sample

(after 24 h) which was 425 higher as compared to controlPoMT2 expression level was upregulated by all the threeconcentrations of CuCl

2after 72 hThe expression of PoMT2

was found to be higher in 40 120583M (1446) and 80120583M (111)treated samples than control In our experiment we havecompared the expression level of MT2 with that of ubiquitinunder similar experimental conditions The result indicatedan enhancement of MT type 2 expression with increasingdoses of copper treatment

Real-time PCR was used for more precise analysis ofexpression pattern of metallothionein type 2 gene Theresults of real-time PCR clearly indicated stress inducedaccumulation of MT2 transcript (Figure 4) At lower dosesthe expression of MT2 slightly increased with respect tocontrol but at higher doses a marked increase in expressionhas been observed Highest expression (49-fold) was foundin 40120583M treated sample However in 80 120583M treated sampleMT2 expression was decreased slightly than 40 120583M dose butstill remained higher (42-fold) than control These resultsare clearly in agreement with the data obtained in reversetranscription PCR Almost similar expression pattern wasreported for MT2 in Arabidopsis [18] In this species MT2amRNA was induced strongly whereas MT2b mRNA levelsincreased only slightly upon exposure to copper [29] Inanother experiment Physcomitrella patens MT2 transcriptwas also induced by copper stress [30] Significant increasein the MT2 transcripts level of Bruguiera gymnorrhiza wasalso found in response to heavy metals like zinc copperand lead [31] The current data suggest that higher doses ofcopper stress upregulate MT2 expression in plant which inturn results in a protection system against this metal

33 Structure Prediction and Phylogenetic Analysis Homol-ogy modeling was used to construct PoMT2 structureThis protein has three significant regions The N-terminal(residues 1ndash23) and C-terminal (residues 67ndash81) Cys richmetal binding domains were fairly conserved with othersequences whereas the spacer region (residues 24ndash66) wasvariable These three regions of PoMT2 were modeledtogether using ModWeb server As the protein data bankdid not contain any plant MT structure with a sequencethat would be sufficiently similar to the complete sequenceof this protein human ADAM22 (Protein Data Bank ID3G5C) was used as template structure 3D structure wasmade with E-value of 0015 and 2500 sequence identityThe final structure was shown with RMSD value 12227A

Sequencing 7

P major MT2P ovata MT2

S lycopersicum MT2BN glutinosa MT2

A deliciosa MT2

C arabica MT1

A thaliana MT2A

B juncea MT22

B rapa pekinensis MT2B rapa MT2

A thaliana MT2B

B juncea MT25B juncea MT23

R communis MT2C arietinum MT2

T repens MT2V faba MT2

M domestica MT2

B juncea MT21

Hyd

ropa

thy

scor

e

16120804

0minus04

minus08

minus12

minus16

10 20 30 40 50 60 70 80

N-terminal cysteinerich domain

C-terminal cysteinerich domain

C-terminal cysteinerich domain

RMSD value 12227

N-terminal cysteinerich domain

a b c d e

a b

c

d

e

Spacer

Chain A

Template PDB ID 36G5C

119864-value 0015

Sequence identity 2500

Figure 5Modeling and analysis of PoMT2 Five distinct motifs of PoMT2 are indicated as a b c d and e and colored black on the 3DmodelHydropathy scores of each amino acid (position 5ndash77) of PoMT2 are shown at corresponding regions Multiple sequence alignment (shadingshowed residues identical for the given position in most sequences) showing relationship between PoMT2 and other plant MTs includingPlantago majorMT2 Solanum lycopersicumMT2B Nicotiana glutinosaMT2 Actinidia deliciosaMT2Malus domesticaMT2 Coffea arabicaMT1Arabidopsis thalianaMT2ABrassica junceaMT22Brassica rapa subsp pekinensisMT2Brassica rapaMT2Arabidopsis thalianaMT2BBrassica juncea MT21 Brassica juncea MT25 Brassica juncea MT23 Ricinus communis MT2 Cicer arietinum MT2 Trifolium repens MT2andVicia fabaMT2 (Uniprot IDs are Q5ZF75 Q40158 Q40396 P43390 O24058 P43396 P25860 P69163 Q39269 P69164 Q38805 P56168P56172 P56170 P30564 Q39459 P43398 and Q41657 resp)

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

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Advances in

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ISRN Biotechnology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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GenomicsInternational Journal of

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Signal TransductionJournal of

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PeptidesInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

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International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 2: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

2 Sequencing

proteins rich in cysteine (Cys or C) residues that bindheavy metals and play a role in buffering the intracellularconcentration of free thiophilic metal ions such as Cu Znand Cd [14] MTs are widely distributed among the animaland plant kingdom MTs are low molecular weight proteinswhich contain two metal-binding cysteine-rich domainslinked by a Cys-free spacer which varies between plant andanimal [12] MTs also play important roles in regulationof metalloenzymes and transcription factors scavenging ofreactive oxygen species metabolism of metallodrugs andalkylating agents response to stress conditions and apop-tosis [15] Classically MTs were grouped into three classesaccording to their sequence similarities [12] Afterward itwas classified into 15 families on the basis of taxonomicrelationships with plant MTs being placed into Family 15[16] All plant MTs (class II) were further distributed intofour types according to the distribution of cysteine residuesin the amino- and carboxy-terminal regions similarities Thespacer region separating these domains in Type 2 MTs ismuch more variable between species [12] In the presentstudy we found that amino acid sequence of Plantagoovata MT2 is very similar to Plantago major MT2 andthe only difference between them was in the spacer regionSeveral plant MT proteins are found in the data base butvery little experimental structural information has yet beenreported so far due to difficulties in protein purificationThe structure is very useful to understand the biologicalfeatures and functions of a protein Therefore theoreticalmethod is needed to predict the three-dimensional structureof proteins

A novel finding of metallothionein type 2 gene fromPlantago ovata is reported here The objective of the presentwork is to investigate the MT2 expression pattern againstcopper toxicity The isolated and molecular characterizedMT2 transcript and its predicted structure contribute toa better understanding of MT2 on the basis of structure-function relationship in an important medicinal plant like Povata

2 Materials and Method

21 Plant Material and Treatment Seeds of Plantago ovatawere collected from Gujarat India The seeds were sterilizedin 10 sodium hypochlorite and washed five times to washoff the excess bleach totally from the seed surface andimbibed in sterilized distilled water Next day the seedswere transferred to agar-sucrose media containing 3 (wv)sucrose (SRL Mumbai India) and 09 (wv) agar (SRLMumbai India) for germination according to the methodas described by Das and Sen Raychaudhuri [17] Eight-day-old seedlings were transferred to liquidMurashige and Skoog(MS)medium (Himedia Mumbai India) supplemented withdifferent concentrations ofCuCl

2(20120583M40 120583M and 80 120583M)

along with control The specified doses have been selectedafter considering the LD

50of copper in P ovataThe LD

50of

copper was found to be 120120583M in case of P ovate and hencea range of lower doses have been selected [18] CuCl

2(copper

(II) Chloride) (Merck Darmstadt Germany) treatment was

carried out for 24 h and 72 h to observe the changes of MTtranscript levels

22 Genomic DNA Extraction Genomic DNA was isolatedfrom seedlings following the standard protocol of Edwards etal [19] Seedlings were crushed gently using DNA extractionbuffer followed by a centrifugation to remove the tissuefragments Supernatant was taken and equal volume ofphenol chloroform (1 1) (SRL Mumbai India) was addedto it After that a centrifugation was carried out and thesupernatant was washed with equal volume of chloroformFinally the clear upper aqueous phase was collected and 3Mammonium acetate and equal volume of isopropanol (SRLMumbai India) were added and mixed DNA was spooledwith glass capillary and washed with 70 ethanol (SRLMumbai India) Subsequently the DNA was air dried anddissolved in sterile triple distilled water The DNA was run in1 agarose gel and spectrophotometrically scanned to checkthe quality and quantity

23 Total RNA Extraction Total RNA was extracted fromseedlings using RNeasy Plant Mini Kit (Qiagen HamburgGermany) according to the manufacturerrsquos protocol All theglass goods were treated with diethyl pyrocarbonate (SigmaSt Louis USA) prior to RNA extraction RNAwas quantifiedspectrophotometrically The extracted RNA was preserved atminus70∘C

24 Primer Designing The primers for P ovata MT2 weredesigned based on published sequences Published sequenceswere taken using Blast search and they were aligned usingClustalW Primers were designed from those regions ofPlantago major MT2 sequence showing best homology withother published sequences The forward and reverse primersof MT2 were of 23 and 22 bases respectively The primerswere designed in such away so that theirmelting temperaturewould be close The sequences of forward and reverse MT2primers were 51015840-TCTTGCTGCAACGGAAACTGTGG-31015840and 51015840-AGTTGTCACCGCACTTGCACCC-31015840 respectivelyUbiquitin was taken as a housekeeping gene [20 21] tonormalize the reverse transcription PCR analysis Thesequences of ubiquitin primers were designed in thesame procedure as described above The sequences offorward and reverse primer of P ovata ubiquitin were51015840-TGAAAACTTTTACAGGCAAGACC-31015840 and 51015840-GAC-GGAGTACCAAATGGAGAGTG-31015840 respectively

25 Polymerase Chain Reaction (PCR) and Reverse Transcrip-tion Polymerase Chain Reaction (RT-PCR) PCR and RT-PCR were carried out using designed primers Taq enzymeTaq buffer and MgCl

2(magnesium chloride) used in PCR

were brought from Genei (Bangalore India) and were usedaccording to themanufacturerrsquos protocolThe thermal profilefor PCR amplification of MT2 was as follows 94∘C for2min followed by 35 cycles at 94∘C for 1min 62∘C for30 sec and 72∘C for 1min 30 sec and a final extension at72∘C for 10min RT-PCR was carried out using One StepRT-PCR Kit (Qiagen Hamburg Germany) according to

Sequencing 3

the manufacturerrsquos protocol with the designed primers Thereverse transcription experiments were carried out usingequal amounts of total RNA (2 120583g) extracted from copperstressed and control seedlings Simultaneously RT-PCR wasalso carried out for ubiquitin in the same conditions of exper-iment This step was essential to estimate the efficiency ofreverse transcription system after 25 cycles of amplificationPCR and RT-PCR was carried out using GeneAmp PCRSystem (Applied Biosystems Carlsbad USA) The thermalprofile for RT-PCR amplification of MT2 was as follows 50∘Cfor 30min 95∘C for 15min followed by 25 cycles at 94∘Cfor 1min 62∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min The RT-PCR cycle forubiquitin was 50∘C for 30min 95∘C for 15min followedby 25 cycles at 94∘C for 1min 57∘C for 1min and 72∘Cfor 1min 30 sec and a final extension at 72∘C for 10minAliquots of 25 120583L of the PCR and RT-PCR products wererun on 15 and 2 agarose gel respectively The gels werestained with ethidium bromide and photographs were takenusing aGelDocumentation System (BioRadHercules USA)All RT-PCR reactions were replicated at least thrice fromthree independent RNA preparations The pixel intensityof the band in gel was quantified using ImageJ softwareThe obtained results were subjected to statistical analysisusing Kyplot and Microsoft Excel All data were expressed asmean plusmn standard error (SE) These data were analyzed by ananalysis of variance (ANOVA) Groupmeans were comparedusing Studentrsquos 119905 test if significance was found in ANOVADifferences of the data were considered significant when 119875 le005

26 Cloning and Sequence Analysis Cloning and SequenceAnalysis of The PCR product of metallothionein type 2 waspurified by PCR clean-up kit (Chromus Biotech BengaluruIndia) according to the manufacturerrsquos protocol TheMetallothionein gene fragment was cloned into the vectorpTZ57RT using the protocol provided in the InsTAclonePCR cloning Kit (Fermentas St Leon-Rot Germany) Ecoli XL 1- blue was used as a host strain After cloning thepurified plasmid it was sequenced using universal primersThe primer sequences are 51015840-GTAAAACGACGGCCAGT-31015840 and 51015840-CAGGAAACAGC- TATGAC-31015840 In case ofubiquitin the PCR product was sequenced using genespecific primers The sequences were analyzed usingseveral programs such as BLASTN BLASTX BLASTPand ClustalW [22 23] The deduced amino acid sequenceof P ovata MT2 was analyzed to detect the N-terminalsorting signal using iPSORT (httpipsorthgcjp) andto determine the pI value and molecular weight usingExPASy (httpwwwexpasychtools) [22] To determinethe hydrophobic nature of the amino acids in the protein ahydropathy plot was created using ExPASy-ProtScale (httpwebexpasyorgprotscale) choosing Kyte amp Doolittle scaledefault parameters and window size 9 The disulfidebond partners in the P ovata MT2 were predictedusing DiANNA server (httpclaviusbcedusimclotelabDiANNAmainhtml) For better estimation of therelationships between P ovata MT2 and other reported

MTs a phylogenetic tree was generated using Phylip369 package (httpevolutiongeneticswashingtoneduphyliphtml) enabling 100 bootstraps Unique metalloth-ionein sequences were selected from UniProtSwiss-Protknowledgebase (httpwwwuniprotorgdocsmetallotxt) [24 25] One sequence from each metallothioneinsubdivision was taken along with all plant MT (Family 15)to generate a significant phylogenetic tree As primers weredesigned from the available closest species Plantago majoramino acid sequences of metallothionein of this specieswere included in the phylogenetic tree Sequence file (Phylipformat) was run using seqboot choosing 100 replicate andall other default parameters The outfile of this programwas run using protpars for parsimony based phylogeneticanalysis keeping the number of jumbles set to 10 and all otherparameters as default the output tree of this programwas runusing consense Finally the unrooted tree was constructedusing njplot (httppbiluniv-lyon1frsoftwarenjplothtml)

27 Expression Analysis Using Real-Time PCR Formetalloth-ionein type 2 gene expression analysis first strand cDNAwassynthesized from total RNA (sim2120583g) of all the treated (for72 h with different concentrations of CuCl

2) samples using

high-capacity RNA-to-cDNA kit (Applied Biosystems USA)according to the manufacturerrsquos instruction Real-time PCRwas carried out in Step One Plus Real-Time PCR thermocy-cler (Applied Biosystems UK) Each reaction contained 2XPower Cyber Green PCR master mix (Applied BiosystemsUK) diluted cDNA and 10 pmol gene specific primers Thesequences of forward and reverse primers used in real-time PCR were 51015840-TCTTGCTGCAACGGAAACTGTGG-31015840and 51015840-AGTTGTCACCGCACTTGCACCC-31015840 respectivelyFollowing thermal conditions have been used heating at95∘C for 10min 40 cycles of denaturation at 95∘C for 15 secannealing and extension at 60∘C for 1min followed by amelt curve analysis to ensure the amplification of only thedesired amplicon A gene encoding P ovata actin was usedas endogenous control The primers used to amplify actinwere 51015840-ATCATGAAGTGTGATGTTGA-31015840 (forward) and51015840-ACCTTAATCTTCATGCTGCC-31015840 (reverse) The relativegene expression was conducted using the 2minusΔΔCT method[26] The data of relative gene expression were analyzed byStep One software version 21 (Applied Biosystems UK) Allthe experiments were performed in triplicate A negativecontrol was included in each reaction The results presentedhere are means of three replicates and the bars indicatestandard deviation Statistical significance of real-time exper-iment results were analyzed by analysis of variance (ANOVA)using Kyplot software

28 Molecular Modeling of P ovata MT2 The deducedamino acid sequence of P ovata MT2 (complete codingregion) was submitted to ModWeb server (httpsmod-basecompbioucsfeduscgimodwebcgi) for comparativeprotein structure modeling choosing default parameters[27] ModWeb performs automated comparative modelingwhich relies on PSI-BLAST IMPALA and MODELLER[28] This server uses ModPipe (version SVNr13401348M)

4 Sequencing

PoMT2 gene (partial CDS)

1

46

100159218

264

309368427486

536

(a)

PoMT2 mRNA (complete CDS)

1

46

91

136

181

226lowast

(b)

Figure 1 Nucleotide and deduced amino acid sequences of PoMT2 Two introns interrupt the coding region at the points marked by arrows(a) Partial coding region encodes a protein of 71 amino acids (GenBankAccession no GU596501 andGU596503) (b) Complete coding regionencodes a protein of 81 amino acids

1

45

90

135

180

P ovata ubiquitin mRNA (partial CDS)

Figure 2 Nucleotide and deduced amino acid sequences of P ovata ubiquitin (GenBank Accession no JQ419758) Partial coding regionencodes a protein of 67 amino acids

as underlying software pipeline Human ADAM22 chainA (Protein Data Bank ID 3G5C) was used as templatestructure (template region 447ndash523) for modeling of Povata MT2 The structure was reconstructed using theDeepViewSwiss-PdbViewer37 [15]

3 Result and Discussion

31 Cloning and Sequence Analysis PCR was carried outusing genomic DNA and a 578 bp fragment was found This

fragment was purified cloned and sequenced (Figure 1(a))On the other hand RT-PCR was carried out using totalRNA and a 214 bp fragment was found which was alsopurified cloned and sequenced (Figure 1(a)) Comparingboth the sequences it was found that the type 2 metal-lothionein gene of P ovata has 3 exons (0ndash63 222ndash308and 515ndash577) and 2 introns (64ndash221 and 309ndash514) in itssequence MT2 of some other plant species also show 3 exonsand 2 introns like Helianthus annuus (GenBank accessionno EF431954) Typha angustifolia (GenBank accession no

Sequencing 5

1 2 3 4 1 2 3 4

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

lowast lowast

lowastlowastlowast

lowastlowast

24 h 72 h

Pixe

l int

ensit

y (A

U)

Pixe

l int

ensit

y (A

U)

(a)

(b)

(c)

(d)

(e)

Figure 3 Results from reverse transcription PCR analysis Upper panel showing RT-PCR band in agarose gel of metallothionein type 2((a)(c)) and ubiquitin ((b)(d)) transcripts after treatment with different concentrations of CuCl

2(0 20 40 and 80 120583M in lane no 1 2 3

and 4 resp) Lower panel showing densitometry graph of corresponding band of MT2 Data represent the mean plusmn SE Asterisks indicatesignificant differences at 119875 lt 005 (lowast) 119875 lt 001 (lowastlowast) or 119875 lt 0001 (lowastlowastlowast) compared to respective controls AU means arbitrary unit

EF543085) and Hordeum vulgare (GenBank accession noJN997433)

After splicing out of the introns P ovata forms a 214 bpMT2 mRNA This sequence was searched for similar othersequences with the help of BLAST tool and many othersequences of metallothionein type 2 were found with 75ndash90 of sequence identity for example Plantago major MT2Avicennia marina MT2 and Ilex paraguariensis (GenBankaccession numbers are AJ843994 AF333385 and JX271039resp) The deduced amino acid sequence was also searchedby BLASTp tool to confirm that the obtained sequence wasof metallothionein type 2 The resultant nucleotide sequencewas aligned with some other reported metallothionein type 2with the help of ClustalW and similarity was found BLASTpanalysis of the deduced amino acid sequence also showedthat the fragment conformed to metallothionein type 2 superfamily Finally it was confirmed that the cloned sequence

was of P ovata metallothionein type 2 The successfullyisolated partial coding regions of MT2 gene and mRNAsequences of P ovata were submitted to GenBank with theaccession numbers GU596501 and GU596503 respectivelyand these were the first sequences of metallothionein fromthis species A later study by our group has sequenced thecomplete coding regions of MT2 from P ovata (PoMT2)(Figure 1(b)) (submitted to GenBank having accession noKC414846) encoding an 81-amino-acid protein with a cal-culated molecular weight of about 81 kDa and a theoreticalpI value of 477 The primer sequences used here were51015840-ATGTCTTGCTGCAACGGAAACT-31015840 (forward) and 51015840-CTATTTGCAATTGCATGGATTG-31015840 (reverse) The ther-mal profile for RT-PCR reaction was as follows 50∘C for30min 95∘C for 15min followed by 25 cycles at 94∘C for1min 54∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min Analysis of this PoMT2

6 Sequencing

6

5

4

3

2

1

0

Nor

mal

ized

relat

ive e

xpre

ssio

n

Control 20 120583M 40 120583M 80 120583MCuCl2 concentration in media

Figure 4 Real-time PCR expression profile of PoMT2 normalizedto the housekeeping gene actin under 0 20 40 and 80120583M copperstress The results indicate means of three replicates with theirstandard deviation

using iPSORT predicted that it does not have any mitochon-drial targeting peptide or chloroplast transit peptide UsingDiANNA server it was found that the PoMT2 has 7 predicteddisulfide bond between 3ndash8 4ndash75 10ndash67 14ndash20 16ndash78 23ndash80and 69ndash73 amino acid positions The terminal domain con-tained 0 aromatic 263 nonpolar aliphatic 605 polarand 105 charged amino acids In case of spacer region thesevalues were 93 419 302 and 186 respectively Thisprotein sequence of PoMT2 was compared using ClustalWand Blastp indicates that PoMT2 has 84 sequence identityto theMT2 encoded by Plantagomajor followed byAvicenniamarina 72 identity and Citrullus lanatus at 70 It hasbeen reported that class I MTs contain 20 highly conservedC residues showing homology to mammalian MTs whereasMTs without this strict arrangement of cysteines are referredto as class II MTs These MTs do not show homology tomammalian MTs Type 2 MTs contain CC CXC and CXXCmotifs in the N-terminal and CXC motif in the C-terminaldomain X can be any amino acid other than cysteine It alsocontains a CGGCmotif at the end of theN-terminal cysteine-rich domain and a spacer of approximately 40 amino acidresidues [12 15] In the present experiment PoMT2 followsall these criteria confirming that the isolated metallothioneinis a class II Type 2 plant metallothionein

The reverse transcription PCR product for ubiquitinwas sequenced A 204 bp sequence was obtained (Figure 2)(encoding 67 amino acids) which was searched for similarother sequences with the help of BLAST tool as describedabove It was found that the cloned sequence showed a goodsimilarity with some other published ubiquitin sequencesFor further confirmation the deduced amino acid sequencewas also searched by BLASTp tool and 99 sequenceidentity was found with other reported ubiquitin sequencesof Arabidopsis thaliana Plantago major Musa acuminateand Medicago truncatula (GenBank accession numbers areAAB95251 CAH56488 AAQ07454 and AET01627 resp)Finally it was submitted to GenBank with the accession

number JQ419758 It was the first ubiquitin sequence of Povata

32 Expression of PoMT2 Transcript in Different Stress Condi-tion To characterize PoMT2 transcript accumulation totalRNA extracted from various stress conditions were subjectedto reverse transcription (RT)-PCR reaction The seedlingswere treated with 0 20 40 and 80 120583M of CuCl

2solutions

for 24 h and 72 h (Figure 3) In each case 25 cycles of PCRreaction with first strand cDNA was carried out All thereactions showed the presence of a 214 bp bandThe intensityof the band was maximum in 40M CuCl

2treated sample

(after 24 h) which was 425 higher as compared to controlPoMT2 expression level was upregulated by all the threeconcentrations of CuCl

2after 72 hThe expression of PoMT2

was found to be higher in 40 120583M (1446) and 80120583M (111)treated samples than control In our experiment we havecompared the expression level of MT2 with that of ubiquitinunder similar experimental conditions The result indicatedan enhancement of MT type 2 expression with increasingdoses of copper treatment

Real-time PCR was used for more precise analysis ofexpression pattern of metallothionein type 2 gene Theresults of real-time PCR clearly indicated stress inducedaccumulation of MT2 transcript (Figure 4) At lower dosesthe expression of MT2 slightly increased with respect tocontrol but at higher doses a marked increase in expressionhas been observed Highest expression (49-fold) was foundin 40120583M treated sample However in 80 120583M treated sampleMT2 expression was decreased slightly than 40 120583M dose butstill remained higher (42-fold) than control These resultsare clearly in agreement with the data obtained in reversetranscription PCR Almost similar expression pattern wasreported for MT2 in Arabidopsis [18] In this species MT2amRNA was induced strongly whereas MT2b mRNA levelsincreased only slightly upon exposure to copper [29] Inanother experiment Physcomitrella patens MT2 transcriptwas also induced by copper stress [30] Significant increasein the MT2 transcripts level of Bruguiera gymnorrhiza wasalso found in response to heavy metals like zinc copperand lead [31] The current data suggest that higher doses ofcopper stress upregulate MT2 expression in plant which inturn results in a protection system against this metal

33 Structure Prediction and Phylogenetic Analysis Homol-ogy modeling was used to construct PoMT2 structureThis protein has three significant regions The N-terminal(residues 1ndash23) and C-terminal (residues 67ndash81) Cys richmetal binding domains were fairly conserved with othersequences whereas the spacer region (residues 24ndash66) wasvariable These three regions of PoMT2 were modeledtogether using ModWeb server As the protein data bankdid not contain any plant MT structure with a sequencethat would be sufficiently similar to the complete sequenceof this protein human ADAM22 (Protein Data Bank ID3G5C) was used as template structure 3D structure wasmade with E-value of 0015 and 2500 sequence identityThe final structure was shown with RMSD value 12227A

Sequencing 7

P major MT2P ovata MT2

S lycopersicum MT2BN glutinosa MT2

A deliciosa MT2

C arabica MT1

A thaliana MT2A

B juncea MT22

B rapa pekinensis MT2B rapa MT2

A thaliana MT2B

B juncea MT25B juncea MT23

R communis MT2C arietinum MT2

T repens MT2V faba MT2

M domestica MT2

B juncea MT21

Hyd

ropa

thy

scor

e

16120804

0minus04

minus08

minus12

minus16

10 20 30 40 50 60 70 80

N-terminal cysteinerich domain

C-terminal cysteinerich domain

C-terminal cysteinerich domain

RMSD value 12227

N-terminal cysteinerich domain

a b c d e

a b

c

d

e

Spacer

Chain A

Template PDB ID 36G5C

119864-value 0015

Sequence identity 2500

Figure 5Modeling and analysis of PoMT2 Five distinct motifs of PoMT2 are indicated as a b c d and e and colored black on the 3DmodelHydropathy scores of each amino acid (position 5ndash77) of PoMT2 are shown at corresponding regions Multiple sequence alignment (shadingshowed residues identical for the given position in most sequences) showing relationship between PoMT2 and other plant MTs includingPlantago majorMT2 Solanum lycopersicumMT2B Nicotiana glutinosaMT2 Actinidia deliciosaMT2Malus domesticaMT2 Coffea arabicaMT1Arabidopsis thalianaMT2ABrassica junceaMT22Brassica rapa subsp pekinensisMT2Brassica rapaMT2Arabidopsis thalianaMT2BBrassica juncea MT21 Brassica juncea MT25 Brassica juncea MT23 Ricinus communis MT2 Cicer arietinum MT2 Trifolium repens MT2andVicia fabaMT2 (Uniprot IDs are Q5ZF75 Q40158 Q40396 P43390 O24058 P43396 P25860 P69163 Q39269 P69164 Q38805 P56168P56172 P56170 P30564 Q39459 P43398 and Q41657 resp)

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

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PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 3: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

Sequencing 3

the manufacturerrsquos protocol with the designed primers Thereverse transcription experiments were carried out usingequal amounts of total RNA (2 120583g) extracted from copperstressed and control seedlings Simultaneously RT-PCR wasalso carried out for ubiquitin in the same conditions of exper-iment This step was essential to estimate the efficiency ofreverse transcription system after 25 cycles of amplificationPCR and RT-PCR was carried out using GeneAmp PCRSystem (Applied Biosystems Carlsbad USA) The thermalprofile for RT-PCR amplification of MT2 was as follows 50∘Cfor 30min 95∘C for 15min followed by 25 cycles at 94∘Cfor 1min 62∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min The RT-PCR cycle forubiquitin was 50∘C for 30min 95∘C for 15min followedby 25 cycles at 94∘C for 1min 57∘C for 1min and 72∘Cfor 1min 30 sec and a final extension at 72∘C for 10minAliquots of 25 120583L of the PCR and RT-PCR products wererun on 15 and 2 agarose gel respectively The gels werestained with ethidium bromide and photographs were takenusing aGelDocumentation System (BioRadHercules USA)All RT-PCR reactions were replicated at least thrice fromthree independent RNA preparations The pixel intensityof the band in gel was quantified using ImageJ softwareThe obtained results were subjected to statistical analysisusing Kyplot and Microsoft Excel All data were expressed asmean plusmn standard error (SE) These data were analyzed by ananalysis of variance (ANOVA) Groupmeans were comparedusing Studentrsquos 119905 test if significance was found in ANOVADifferences of the data were considered significant when 119875 le005

26 Cloning and Sequence Analysis Cloning and SequenceAnalysis of The PCR product of metallothionein type 2 waspurified by PCR clean-up kit (Chromus Biotech BengaluruIndia) according to the manufacturerrsquos protocol TheMetallothionein gene fragment was cloned into the vectorpTZ57RT using the protocol provided in the InsTAclonePCR cloning Kit (Fermentas St Leon-Rot Germany) Ecoli XL 1- blue was used as a host strain After cloning thepurified plasmid it was sequenced using universal primersThe primer sequences are 51015840-GTAAAACGACGGCCAGT-31015840 and 51015840-CAGGAAACAGC- TATGAC-31015840 In case ofubiquitin the PCR product was sequenced using genespecific primers The sequences were analyzed usingseveral programs such as BLASTN BLASTX BLASTPand ClustalW [22 23] The deduced amino acid sequenceof P ovata MT2 was analyzed to detect the N-terminalsorting signal using iPSORT (httpipsorthgcjp) andto determine the pI value and molecular weight usingExPASy (httpwwwexpasychtools) [22] To determinethe hydrophobic nature of the amino acids in the protein ahydropathy plot was created using ExPASy-ProtScale (httpwebexpasyorgprotscale) choosing Kyte amp Doolittle scaledefault parameters and window size 9 The disulfidebond partners in the P ovata MT2 were predictedusing DiANNA server (httpclaviusbcedusimclotelabDiANNAmainhtml) For better estimation of therelationships between P ovata MT2 and other reported

MTs a phylogenetic tree was generated using Phylip369 package (httpevolutiongeneticswashingtoneduphyliphtml) enabling 100 bootstraps Unique metalloth-ionein sequences were selected from UniProtSwiss-Protknowledgebase (httpwwwuniprotorgdocsmetallotxt) [24 25] One sequence from each metallothioneinsubdivision was taken along with all plant MT (Family 15)to generate a significant phylogenetic tree As primers weredesigned from the available closest species Plantago majoramino acid sequences of metallothionein of this specieswere included in the phylogenetic tree Sequence file (Phylipformat) was run using seqboot choosing 100 replicate andall other default parameters The outfile of this programwas run using protpars for parsimony based phylogeneticanalysis keeping the number of jumbles set to 10 and all otherparameters as default the output tree of this programwas runusing consense Finally the unrooted tree was constructedusing njplot (httppbiluniv-lyon1frsoftwarenjplothtml)

27 Expression Analysis Using Real-Time PCR Formetalloth-ionein type 2 gene expression analysis first strand cDNAwassynthesized from total RNA (sim2120583g) of all the treated (for72 h with different concentrations of CuCl

2) samples using

high-capacity RNA-to-cDNA kit (Applied Biosystems USA)according to the manufacturerrsquos instruction Real-time PCRwas carried out in Step One Plus Real-Time PCR thermocy-cler (Applied Biosystems UK) Each reaction contained 2XPower Cyber Green PCR master mix (Applied BiosystemsUK) diluted cDNA and 10 pmol gene specific primers Thesequences of forward and reverse primers used in real-time PCR were 51015840-TCTTGCTGCAACGGAAACTGTGG-31015840and 51015840-AGTTGTCACCGCACTTGCACCC-31015840 respectivelyFollowing thermal conditions have been used heating at95∘C for 10min 40 cycles of denaturation at 95∘C for 15 secannealing and extension at 60∘C for 1min followed by amelt curve analysis to ensure the amplification of only thedesired amplicon A gene encoding P ovata actin was usedas endogenous control The primers used to amplify actinwere 51015840-ATCATGAAGTGTGATGTTGA-31015840 (forward) and51015840-ACCTTAATCTTCATGCTGCC-31015840 (reverse) The relativegene expression was conducted using the 2minusΔΔCT method[26] The data of relative gene expression were analyzed byStep One software version 21 (Applied Biosystems UK) Allthe experiments were performed in triplicate A negativecontrol was included in each reaction The results presentedhere are means of three replicates and the bars indicatestandard deviation Statistical significance of real-time exper-iment results were analyzed by analysis of variance (ANOVA)using Kyplot software

28 Molecular Modeling of P ovata MT2 The deducedamino acid sequence of P ovata MT2 (complete codingregion) was submitted to ModWeb server (httpsmod-basecompbioucsfeduscgimodwebcgi) for comparativeprotein structure modeling choosing default parameters[27] ModWeb performs automated comparative modelingwhich relies on PSI-BLAST IMPALA and MODELLER[28] This server uses ModPipe (version SVNr13401348M)

4 Sequencing

PoMT2 gene (partial CDS)

1

46

100159218

264

309368427486

536

(a)

PoMT2 mRNA (complete CDS)

1

46

91

136

181

226lowast

(b)

Figure 1 Nucleotide and deduced amino acid sequences of PoMT2 Two introns interrupt the coding region at the points marked by arrows(a) Partial coding region encodes a protein of 71 amino acids (GenBankAccession no GU596501 andGU596503) (b) Complete coding regionencodes a protein of 81 amino acids

1

45

90

135

180

P ovata ubiquitin mRNA (partial CDS)

Figure 2 Nucleotide and deduced amino acid sequences of P ovata ubiquitin (GenBank Accession no JQ419758) Partial coding regionencodes a protein of 67 amino acids

as underlying software pipeline Human ADAM22 chainA (Protein Data Bank ID 3G5C) was used as templatestructure (template region 447ndash523) for modeling of Povata MT2 The structure was reconstructed using theDeepViewSwiss-PdbViewer37 [15]

3 Result and Discussion

31 Cloning and Sequence Analysis PCR was carried outusing genomic DNA and a 578 bp fragment was found This

fragment was purified cloned and sequenced (Figure 1(a))On the other hand RT-PCR was carried out using totalRNA and a 214 bp fragment was found which was alsopurified cloned and sequenced (Figure 1(a)) Comparingboth the sequences it was found that the type 2 metal-lothionein gene of P ovata has 3 exons (0ndash63 222ndash308and 515ndash577) and 2 introns (64ndash221 and 309ndash514) in itssequence MT2 of some other plant species also show 3 exonsand 2 introns like Helianthus annuus (GenBank accessionno EF431954) Typha angustifolia (GenBank accession no

Sequencing 5

1 2 3 4 1 2 3 4

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

lowast lowast

lowastlowastlowast

lowastlowast

24 h 72 h

Pixe

l int

ensit

y (A

U)

Pixe

l int

ensit

y (A

U)

(a)

(b)

(c)

(d)

(e)

Figure 3 Results from reverse transcription PCR analysis Upper panel showing RT-PCR band in agarose gel of metallothionein type 2((a)(c)) and ubiquitin ((b)(d)) transcripts after treatment with different concentrations of CuCl

2(0 20 40 and 80 120583M in lane no 1 2 3

and 4 resp) Lower panel showing densitometry graph of corresponding band of MT2 Data represent the mean plusmn SE Asterisks indicatesignificant differences at 119875 lt 005 (lowast) 119875 lt 001 (lowastlowast) or 119875 lt 0001 (lowastlowastlowast) compared to respective controls AU means arbitrary unit

EF543085) and Hordeum vulgare (GenBank accession noJN997433)

After splicing out of the introns P ovata forms a 214 bpMT2 mRNA This sequence was searched for similar othersequences with the help of BLAST tool and many othersequences of metallothionein type 2 were found with 75ndash90 of sequence identity for example Plantago major MT2Avicennia marina MT2 and Ilex paraguariensis (GenBankaccession numbers are AJ843994 AF333385 and JX271039resp) The deduced amino acid sequence was also searchedby BLASTp tool to confirm that the obtained sequence wasof metallothionein type 2 The resultant nucleotide sequencewas aligned with some other reported metallothionein type 2with the help of ClustalW and similarity was found BLASTpanalysis of the deduced amino acid sequence also showedthat the fragment conformed to metallothionein type 2 superfamily Finally it was confirmed that the cloned sequence

was of P ovata metallothionein type 2 The successfullyisolated partial coding regions of MT2 gene and mRNAsequences of P ovata were submitted to GenBank with theaccession numbers GU596501 and GU596503 respectivelyand these were the first sequences of metallothionein fromthis species A later study by our group has sequenced thecomplete coding regions of MT2 from P ovata (PoMT2)(Figure 1(b)) (submitted to GenBank having accession noKC414846) encoding an 81-amino-acid protein with a cal-culated molecular weight of about 81 kDa and a theoreticalpI value of 477 The primer sequences used here were51015840-ATGTCTTGCTGCAACGGAAACT-31015840 (forward) and 51015840-CTATTTGCAATTGCATGGATTG-31015840 (reverse) The ther-mal profile for RT-PCR reaction was as follows 50∘C for30min 95∘C for 15min followed by 25 cycles at 94∘C for1min 54∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min Analysis of this PoMT2

6 Sequencing

6

5

4

3

2

1

0

Nor

mal

ized

relat

ive e

xpre

ssio

n

Control 20 120583M 40 120583M 80 120583MCuCl2 concentration in media

Figure 4 Real-time PCR expression profile of PoMT2 normalizedto the housekeeping gene actin under 0 20 40 and 80120583M copperstress The results indicate means of three replicates with theirstandard deviation

using iPSORT predicted that it does not have any mitochon-drial targeting peptide or chloroplast transit peptide UsingDiANNA server it was found that the PoMT2 has 7 predicteddisulfide bond between 3ndash8 4ndash75 10ndash67 14ndash20 16ndash78 23ndash80and 69ndash73 amino acid positions The terminal domain con-tained 0 aromatic 263 nonpolar aliphatic 605 polarand 105 charged amino acids In case of spacer region thesevalues were 93 419 302 and 186 respectively Thisprotein sequence of PoMT2 was compared using ClustalWand Blastp indicates that PoMT2 has 84 sequence identityto theMT2 encoded by Plantagomajor followed byAvicenniamarina 72 identity and Citrullus lanatus at 70 It hasbeen reported that class I MTs contain 20 highly conservedC residues showing homology to mammalian MTs whereasMTs without this strict arrangement of cysteines are referredto as class II MTs These MTs do not show homology tomammalian MTs Type 2 MTs contain CC CXC and CXXCmotifs in the N-terminal and CXC motif in the C-terminaldomain X can be any amino acid other than cysteine It alsocontains a CGGCmotif at the end of theN-terminal cysteine-rich domain and a spacer of approximately 40 amino acidresidues [12 15] In the present experiment PoMT2 followsall these criteria confirming that the isolated metallothioneinis a class II Type 2 plant metallothionein

The reverse transcription PCR product for ubiquitinwas sequenced A 204 bp sequence was obtained (Figure 2)(encoding 67 amino acids) which was searched for similarother sequences with the help of BLAST tool as describedabove It was found that the cloned sequence showed a goodsimilarity with some other published ubiquitin sequencesFor further confirmation the deduced amino acid sequencewas also searched by BLASTp tool and 99 sequenceidentity was found with other reported ubiquitin sequencesof Arabidopsis thaliana Plantago major Musa acuminateand Medicago truncatula (GenBank accession numbers areAAB95251 CAH56488 AAQ07454 and AET01627 resp)Finally it was submitted to GenBank with the accession

number JQ419758 It was the first ubiquitin sequence of Povata

32 Expression of PoMT2 Transcript in Different Stress Condi-tion To characterize PoMT2 transcript accumulation totalRNA extracted from various stress conditions were subjectedto reverse transcription (RT)-PCR reaction The seedlingswere treated with 0 20 40 and 80 120583M of CuCl

2solutions

for 24 h and 72 h (Figure 3) In each case 25 cycles of PCRreaction with first strand cDNA was carried out All thereactions showed the presence of a 214 bp bandThe intensityof the band was maximum in 40M CuCl

2treated sample

(after 24 h) which was 425 higher as compared to controlPoMT2 expression level was upregulated by all the threeconcentrations of CuCl

2after 72 hThe expression of PoMT2

was found to be higher in 40 120583M (1446) and 80120583M (111)treated samples than control In our experiment we havecompared the expression level of MT2 with that of ubiquitinunder similar experimental conditions The result indicatedan enhancement of MT type 2 expression with increasingdoses of copper treatment

Real-time PCR was used for more precise analysis ofexpression pattern of metallothionein type 2 gene Theresults of real-time PCR clearly indicated stress inducedaccumulation of MT2 transcript (Figure 4) At lower dosesthe expression of MT2 slightly increased with respect tocontrol but at higher doses a marked increase in expressionhas been observed Highest expression (49-fold) was foundin 40120583M treated sample However in 80 120583M treated sampleMT2 expression was decreased slightly than 40 120583M dose butstill remained higher (42-fold) than control These resultsare clearly in agreement with the data obtained in reversetranscription PCR Almost similar expression pattern wasreported for MT2 in Arabidopsis [18] In this species MT2amRNA was induced strongly whereas MT2b mRNA levelsincreased only slightly upon exposure to copper [29] Inanother experiment Physcomitrella patens MT2 transcriptwas also induced by copper stress [30] Significant increasein the MT2 transcripts level of Bruguiera gymnorrhiza wasalso found in response to heavy metals like zinc copperand lead [31] The current data suggest that higher doses ofcopper stress upregulate MT2 expression in plant which inturn results in a protection system against this metal

33 Structure Prediction and Phylogenetic Analysis Homol-ogy modeling was used to construct PoMT2 structureThis protein has three significant regions The N-terminal(residues 1ndash23) and C-terminal (residues 67ndash81) Cys richmetal binding domains were fairly conserved with othersequences whereas the spacer region (residues 24ndash66) wasvariable These three regions of PoMT2 were modeledtogether using ModWeb server As the protein data bankdid not contain any plant MT structure with a sequencethat would be sufficiently similar to the complete sequenceof this protein human ADAM22 (Protein Data Bank ID3G5C) was used as template structure 3D structure wasmade with E-value of 0015 and 2500 sequence identityThe final structure was shown with RMSD value 12227A

Sequencing 7

P major MT2P ovata MT2

S lycopersicum MT2BN glutinosa MT2

A deliciosa MT2

C arabica MT1

A thaliana MT2A

B juncea MT22

B rapa pekinensis MT2B rapa MT2

A thaliana MT2B

B juncea MT25B juncea MT23

R communis MT2C arietinum MT2

T repens MT2V faba MT2

M domestica MT2

B juncea MT21

Hyd

ropa

thy

scor

e

16120804

0minus04

minus08

minus12

minus16

10 20 30 40 50 60 70 80

N-terminal cysteinerich domain

C-terminal cysteinerich domain

C-terminal cysteinerich domain

RMSD value 12227

N-terminal cysteinerich domain

a b c d e

a b

c

d

e

Spacer

Chain A

Template PDB ID 36G5C

119864-value 0015

Sequence identity 2500

Figure 5Modeling and analysis of PoMT2 Five distinct motifs of PoMT2 are indicated as a b c d and e and colored black on the 3DmodelHydropathy scores of each amino acid (position 5ndash77) of PoMT2 are shown at corresponding regions Multiple sequence alignment (shadingshowed residues identical for the given position in most sequences) showing relationship between PoMT2 and other plant MTs includingPlantago majorMT2 Solanum lycopersicumMT2B Nicotiana glutinosaMT2 Actinidia deliciosaMT2Malus domesticaMT2 Coffea arabicaMT1Arabidopsis thalianaMT2ABrassica junceaMT22Brassica rapa subsp pekinensisMT2Brassica rapaMT2Arabidopsis thalianaMT2BBrassica juncea MT21 Brassica juncea MT25 Brassica juncea MT23 Ricinus communis MT2 Cicer arietinum MT2 Trifolium repens MT2andVicia fabaMT2 (Uniprot IDs are Q5ZF75 Q40158 Q40396 P43390 O24058 P43396 P25860 P69163 Q39269 P69164 Q38805 P56168P56172 P56170 P30564 Q39459 P43398 and Q41657 resp)

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

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PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

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International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 4: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

4 Sequencing

PoMT2 gene (partial CDS)

1

46

100159218

264

309368427486

536

(a)

PoMT2 mRNA (complete CDS)

1

46

91

136

181

226lowast

(b)

Figure 1 Nucleotide and deduced amino acid sequences of PoMT2 Two introns interrupt the coding region at the points marked by arrows(a) Partial coding region encodes a protein of 71 amino acids (GenBankAccession no GU596501 andGU596503) (b) Complete coding regionencodes a protein of 81 amino acids

1

45

90

135

180

P ovata ubiquitin mRNA (partial CDS)

Figure 2 Nucleotide and deduced amino acid sequences of P ovata ubiquitin (GenBank Accession no JQ419758) Partial coding regionencodes a protein of 67 amino acids

as underlying software pipeline Human ADAM22 chainA (Protein Data Bank ID 3G5C) was used as templatestructure (template region 447ndash523) for modeling of Povata MT2 The structure was reconstructed using theDeepViewSwiss-PdbViewer37 [15]

3 Result and Discussion

31 Cloning and Sequence Analysis PCR was carried outusing genomic DNA and a 578 bp fragment was found This

fragment was purified cloned and sequenced (Figure 1(a))On the other hand RT-PCR was carried out using totalRNA and a 214 bp fragment was found which was alsopurified cloned and sequenced (Figure 1(a)) Comparingboth the sequences it was found that the type 2 metal-lothionein gene of P ovata has 3 exons (0ndash63 222ndash308and 515ndash577) and 2 introns (64ndash221 and 309ndash514) in itssequence MT2 of some other plant species also show 3 exonsand 2 introns like Helianthus annuus (GenBank accessionno EF431954) Typha angustifolia (GenBank accession no

Sequencing 5

1 2 3 4 1 2 3 4

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

lowast lowast

lowastlowastlowast

lowastlowast

24 h 72 h

Pixe

l int

ensit

y (A

U)

Pixe

l int

ensit

y (A

U)

(a)

(b)

(c)

(d)

(e)

Figure 3 Results from reverse transcription PCR analysis Upper panel showing RT-PCR band in agarose gel of metallothionein type 2((a)(c)) and ubiquitin ((b)(d)) transcripts after treatment with different concentrations of CuCl

2(0 20 40 and 80 120583M in lane no 1 2 3

and 4 resp) Lower panel showing densitometry graph of corresponding band of MT2 Data represent the mean plusmn SE Asterisks indicatesignificant differences at 119875 lt 005 (lowast) 119875 lt 001 (lowastlowast) or 119875 lt 0001 (lowastlowastlowast) compared to respective controls AU means arbitrary unit

EF543085) and Hordeum vulgare (GenBank accession noJN997433)

After splicing out of the introns P ovata forms a 214 bpMT2 mRNA This sequence was searched for similar othersequences with the help of BLAST tool and many othersequences of metallothionein type 2 were found with 75ndash90 of sequence identity for example Plantago major MT2Avicennia marina MT2 and Ilex paraguariensis (GenBankaccession numbers are AJ843994 AF333385 and JX271039resp) The deduced amino acid sequence was also searchedby BLASTp tool to confirm that the obtained sequence wasof metallothionein type 2 The resultant nucleotide sequencewas aligned with some other reported metallothionein type 2with the help of ClustalW and similarity was found BLASTpanalysis of the deduced amino acid sequence also showedthat the fragment conformed to metallothionein type 2 superfamily Finally it was confirmed that the cloned sequence

was of P ovata metallothionein type 2 The successfullyisolated partial coding regions of MT2 gene and mRNAsequences of P ovata were submitted to GenBank with theaccession numbers GU596501 and GU596503 respectivelyand these were the first sequences of metallothionein fromthis species A later study by our group has sequenced thecomplete coding regions of MT2 from P ovata (PoMT2)(Figure 1(b)) (submitted to GenBank having accession noKC414846) encoding an 81-amino-acid protein with a cal-culated molecular weight of about 81 kDa and a theoreticalpI value of 477 The primer sequences used here were51015840-ATGTCTTGCTGCAACGGAAACT-31015840 (forward) and 51015840-CTATTTGCAATTGCATGGATTG-31015840 (reverse) The ther-mal profile for RT-PCR reaction was as follows 50∘C for30min 95∘C for 15min followed by 25 cycles at 94∘C for1min 54∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min Analysis of this PoMT2

6 Sequencing

6

5

4

3

2

1

0

Nor

mal

ized

relat

ive e

xpre

ssio

n

Control 20 120583M 40 120583M 80 120583MCuCl2 concentration in media

Figure 4 Real-time PCR expression profile of PoMT2 normalizedto the housekeeping gene actin under 0 20 40 and 80120583M copperstress The results indicate means of three replicates with theirstandard deviation

using iPSORT predicted that it does not have any mitochon-drial targeting peptide or chloroplast transit peptide UsingDiANNA server it was found that the PoMT2 has 7 predicteddisulfide bond between 3ndash8 4ndash75 10ndash67 14ndash20 16ndash78 23ndash80and 69ndash73 amino acid positions The terminal domain con-tained 0 aromatic 263 nonpolar aliphatic 605 polarand 105 charged amino acids In case of spacer region thesevalues were 93 419 302 and 186 respectively Thisprotein sequence of PoMT2 was compared using ClustalWand Blastp indicates that PoMT2 has 84 sequence identityto theMT2 encoded by Plantagomajor followed byAvicenniamarina 72 identity and Citrullus lanatus at 70 It hasbeen reported that class I MTs contain 20 highly conservedC residues showing homology to mammalian MTs whereasMTs without this strict arrangement of cysteines are referredto as class II MTs These MTs do not show homology tomammalian MTs Type 2 MTs contain CC CXC and CXXCmotifs in the N-terminal and CXC motif in the C-terminaldomain X can be any amino acid other than cysteine It alsocontains a CGGCmotif at the end of theN-terminal cysteine-rich domain and a spacer of approximately 40 amino acidresidues [12 15] In the present experiment PoMT2 followsall these criteria confirming that the isolated metallothioneinis a class II Type 2 plant metallothionein

The reverse transcription PCR product for ubiquitinwas sequenced A 204 bp sequence was obtained (Figure 2)(encoding 67 amino acids) which was searched for similarother sequences with the help of BLAST tool as describedabove It was found that the cloned sequence showed a goodsimilarity with some other published ubiquitin sequencesFor further confirmation the deduced amino acid sequencewas also searched by BLASTp tool and 99 sequenceidentity was found with other reported ubiquitin sequencesof Arabidopsis thaliana Plantago major Musa acuminateand Medicago truncatula (GenBank accession numbers areAAB95251 CAH56488 AAQ07454 and AET01627 resp)Finally it was submitted to GenBank with the accession

number JQ419758 It was the first ubiquitin sequence of Povata

32 Expression of PoMT2 Transcript in Different Stress Condi-tion To characterize PoMT2 transcript accumulation totalRNA extracted from various stress conditions were subjectedto reverse transcription (RT)-PCR reaction The seedlingswere treated with 0 20 40 and 80 120583M of CuCl

2solutions

for 24 h and 72 h (Figure 3) In each case 25 cycles of PCRreaction with first strand cDNA was carried out All thereactions showed the presence of a 214 bp bandThe intensityof the band was maximum in 40M CuCl

2treated sample

(after 24 h) which was 425 higher as compared to controlPoMT2 expression level was upregulated by all the threeconcentrations of CuCl

2after 72 hThe expression of PoMT2

was found to be higher in 40 120583M (1446) and 80120583M (111)treated samples than control In our experiment we havecompared the expression level of MT2 with that of ubiquitinunder similar experimental conditions The result indicatedan enhancement of MT type 2 expression with increasingdoses of copper treatment

Real-time PCR was used for more precise analysis ofexpression pattern of metallothionein type 2 gene Theresults of real-time PCR clearly indicated stress inducedaccumulation of MT2 transcript (Figure 4) At lower dosesthe expression of MT2 slightly increased with respect tocontrol but at higher doses a marked increase in expressionhas been observed Highest expression (49-fold) was foundin 40120583M treated sample However in 80 120583M treated sampleMT2 expression was decreased slightly than 40 120583M dose butstill remained higher (42-fold) than control These resultsare clearly in agreement with the data obtained in reversetranscription PCR Almost similar expression pattern wasreported for MT2 in Arabidopsis [18] In this species MT2amRNA was induced strongly whereas MT2b mRNA levelsincreased only slightly upon exposure to copper [29] Inanother experiment Physcomitrella patens MT2 transcriptwas also induced by copper stress [30] Significant increasein the MT2 transcripts level of Bruguiera gymnorrhiza wasalso found in response to heavy metals like zinc copperand lead [31] The current data suggest that higher doses ofcopper stress upregulate MT2 expression in plant which inturn results in a protection system against this metal

33 Structure Prediction and Phylogenetic Analysis Homol-ogy modeling was used to construct PoMT2 structureThis protein has three significant regions The N-terminal(residues 1ndash23) and C-terminal (residues 67ndash81) Cys richmetal binding domains were fairly conserved with othersequences whereas the spacer region (residues 24ndash66) wasvariable These three regions of PoMT2 were modeledtogether using ModWeb server As the protein data bankdid not contain any plant MT structure with a sequencethat would be sufficiently similar to the complete sequenceof this protein human ADAM22 (Protein Data Bank ID3G5C) was used as template structure 3D structure wasmade with E-value of 0015 and 2500 sequence identityThe final structure was shown with RMSD value 12227A

Sequencing 7

P major MT2P ovata MT2

S lycopersicum MT2BN glutinosa MT2

A deliciosa MT2

C arabica MT1

A thaliana MT2A

B juncea MT22

B rapa pekinensis MT2B rapa MT2

A thaliana MT2B

B juncea MT25B juncea MT23

R communis MT2C arietinum MT2

T repens MT2V faba MT2

M domestica MT2

B juncea MT21

Hyd

ropa

thy

scor

e

16120804

0minus04

minus08

minus12

minus16

10 20 30 40 50 60 70 80

N-terminal cysteinerich domain

C-terminal cysteinerich domain

C-terminal cysteinerich domain

RMSD value 12227

N-terminal cysteinerich domain

a b c d e

a b

c

d

e

Spacer

Chain A

Template PDB ID 36G5C

119864-value 0015

Sequence identity 2500

Figure 5Modeling and analysis of PoMT2 Five distinct motifs of PoMT2 are indicated as a b c d and e and colored black on the 3DmodelHydropathy scores of each amino acid (position 5ndash77) of PoMT2 are shown at corresponding regions Multiple sequence alignment (shadingshowed residues identical for the given position in most sequences) showing relationship between PoMT2 and other plant MTs includingPlantago majorMT2 Solanum lycopersicumMT2B Nicotiana glutinosaMT2 Actinidia deliciosaMT2Malus domesticaMT2 Coffea arabicaMT1Arabidopsis thalianaMT2ABrassica junceaMT22Brassica rapa subsp pekinensisMT2Brassica rapaMT2Arabidopsis thalianaMT2BBrassica juncea MT21 Brassica juncea MT25 Brassica juncea MT23 Ricinus communis MT2 Cicer arietinum MT2 Trifolium repens MT2andVicia fabaMT2 (Uniprot IDs are Q5ZF75 Q40158 Q40396 P43390 O24058 P43396 P25860 P69163 Q39269 P69164 Q38805 P56168P56172 P56170 P30564 Q39459 P43398 and Q41657 resp)

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

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GenomicsInternational Journal of

Volume 2013

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PeptidesInternational Journal of

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Stem CellsInternational

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

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The Scientific World Journal

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International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 5: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

Sequencing 5

1 2 3 4 1 2 3 4

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

120

100

80

60

40

20

00 20 40 80

CuCl2 concentration in media (120583M)

lowast lowast

lowastlowastlowast

lowastlowast

24 h 72 h

Pixe

l int

ensit

y (A

U)

Pixe

l int

ensit

y (A

U)

(a)

(b)

(c)

(d)

(e)

Figure 3 Results from reverse transcription PCR analysis Upper panel showing RT-PCR band in agarose gel of metallothionein type 2((a)(c)) and ubiquitin ((b)(d)) transcripts after treatment with different concentrations of CuCl

2(0 20 40 and 80 120583M in lane no 1 2 3

and 4 resp) Lower panel showing densitometry graph of corresponding band of MT2 Data represent the mean plusmn SE Asterisks indicatesignificant differences at 119875 lt 005 (lowast) 119875 lt 001 (lowastlowast) or 119875 lt 0001 (lowastlowastlowast) compared to respective controls AU means arbitrary unit

EF543085) and Hordeum vulgare (GenBank accession noJN997433)

After splicing out of the introns P ovata forms a 214 bpMT2 mRNA This sequence was searched for similar othersequences with the help of BLAST tool and many othersequences of metallothionein type 2 were found with 75ndash90 of sequence identity for example Plantago major MT2Avicennia marina MT2 and Ilex paraguariensis (GenBankaccession numbers are AJ843994 AF333385 and JX271039resp) The deduced amino acid sequence was also searchedby BLASTp tool to confirm that the obtained sequence wasof metallothionein type 2 The resultant nucleotide sequencewas aligned with some other reported metallothionein type 2with the help of ClustalW and similarity was found BLASTpanalysis of the deduced amino acid sequence also showedthat the fragment conformed to metallothionein type 2 superfamily Finally it was confirmed that the cloned sequence

was of P ovata metallothionein type 2 The successfullyisolated partial coding regions of MT2 gene and mRNAsequences of P ovata were submitted to GenBank with theaccession numbers GU596501 and GU596503 respectivelyand these were the first sequences of metallothionein fromthis species A later study by our group has sequenced thecomplete coding regions of MT2 from P ovata (PoMT2)(Figure 1(b)) (submitted to GenBank having accession noKC414846) encoding an 81-amino-acid protein with a cal-culated molecular weight of about 81 kDa and a theoreticalpI value of 477 The primer sequences used here were51015840-ATGTCTTGCTGCAACGGAAACT-31015840 (forward) and 51015840-CTATTTGCAATTGCATGGATTG-31015840 (reverse) The ther-mal profile for RT-PCR reaction was as follows 50∘C for30min 95∘C for 15min followed by 25 cycles at 94∘C for1min 54∘C for 30 sec and 72∘C for 1min 30 sec and afinal extension at 72∘C for 10min Analysis of this PoMT2

6 Sequencing

6

5

4

3

2

1

0

Nor

mal

ized

relat

ive e

xpre

ssio

n

Control 20 120583M 40 120583M 80 120583MCuCl2 concentration in media

Figure 4 Real-time PCR expression profile of PoMT2 normalizedto the housekeeping gene actin under 0 20 40 and 80120583M copperstress The results indicate means of three replicates with theirstandard deviation

using iPSORT predicted that it does not have any mitochon-drial targeting peptide or chloroplast transit peptide UsingDiANNA server it was found that the PoMT2 has 7 predicteddisulfide bond between 3ndash8 4ndash75 10ndash67 14ndash20 16ndash78 23ndash80and 69ndash73 amino acid positions The terminal domain con-tained 0 aromatic 263 nonpolar aliphatic 605 polarand 105 charged amino acids In case of spacer region thesevalues were 93 419 302 and 186 respectively Thisprotein sequence of PoMT2 was compared using ClustalWand Blastp indicates that PoMT2 has 84 sequence identityto theMT2 encoded by Plantagomajor followed byAvicenniamarina 72 identity and Citrullus lanatus at 70 It hasbeen reported that class I MTs contain 20 highly conservedC residues showing homology to mammalian MTs whereasMTs without this strict arrangement of cysteines are referredto as class II MTs These MTs do not show homology tomammalian MTs Type 2 MTs contain CC CXC and CXXCmotifs in the N-terminal and CXC motif in the C-terminaldomain X can be any amino acid other than cysteine It alsocontains a CGGCmotif at the end of theN-terminal cysteine-rich domain and a spacer of approximately 40 amino acidresidues [12 15] In the present experiment PoMT2 followsall these criteria confirming that the isolated metallothioneinis a class II Type 2 plant metallothionein

The reverse transcription PCR product for ubiquitinwas sequenced A 204 bp sequence was obtained (Figure 2)(encoding 67 amino acids) which was searched for similarother sequences with the help of BLAST tool as describedabove It was found that the cloned sequence showed a goodsimilarity with some other published ubiquitin sequencesFor further confirmation the deduced amino acid sequencewas also searched by BLASTp tool and 99 sequenceidentity was found with other reported ubiquitin sequencesof Arabidopsis thaliana Plantago major Musa acuminateand Medicago truncatula (GenBank accession numbers areAAB95251 CAH56488 AAQ07454 and AET01627 resp)Finally it was submitted to GenBank with the accession

number JQ419758 It was the first ubiquitin sequence of Povata

32 Expression of PoMT2 Transcript in Different Stress Condi-tion To characterize PoMT2 transcript accumulation totalRNA extracted from various stress conditions were subjectedto reverse transcription (RT)-PCR reaction The seedlingswere treated with 0 20 40 and 80 120583M of CuCl

2solutions

for 24 h and 72 h (Figure 3) In each case 25 cycles of PCRreaction with first strand cDNA was carried out All thereactions showed the presence of a 214 bp bandThe intensityof the band was maximum in 40M CuCl

2treated sample

(after 24 h) which was 425 higher as compared to controlPoMT2 expression level was upregulated by all the threeconcentrations of CuCl

2after 72 hThe expression of PoMT2

was found to be higher in 40 120583M (1446) and 80120583M (111)treated samples than control In our experiment we havecompared the expression level of MT2 with that of ubiquitinunder similar experimental conditions The result indicatedan enhancement of MT type 2 expression with increasingdoses of copper treatment

Real-time PCR was used for more precise analysis ofexpression pattern of metallothionein type 2 gene Theresults of real-time PCR clearly indicated stress inducedaccumulation of MT2 transcript (Figure 4) At lower dosesthe expression of MT2 slightly increased with respect tocontrol but at higher doses a marked increase in expressionhas been observed Highest expression (49-fold) was foundin 40120583M treated sample However in 80 120583M treated sampleMT2 expression was decreased slightly than 40 120583M dose butstill remained higher (42-fold) than control These resultsare clearly in agreement with the data obtained in reversetranscription PCR Almost similar expression pattern wasreported for MT2 in Arabidopsis [18] In this species MT2amRNA was induced strongly whereas MT2b mRNA levelsincreased only slightly upon exposure to copper [29] Inanother experiment Physcomitrella patens MT2 transcriptwas also induced by copper stress [30] Significant increasein the MT2 transcripts level of Bruguiera gymnorrhiza wasalso found in response to heavy metals like zinc copperand lead [31] The current data suggest that higher doses ofcopper stress upregulate MT2 expression in plant which inturn results in a protection system against this metal

33 Structure Prediction and Phylogenetic Analysis Homol-ogy modeling was used to construct PoMT2 structureThis protein has three significant regions The N-terminal(residues 1ndash23) and C-terminal (residues 67ndash81) Cys richmetal binding domains were fairly conserved with othersequences whereas the spacer region (residues 24ndash66) wasvariable These three regions of PoMT2 were modeledtogether using ModWeb server As the protein data bankdid not contain any plant MT structure with a sequencethat would be sufficiently similar to the complete sequenceof this protein human ADAM22 (Protein Data Bank ID3G5C) was used as template structure 3D structure wasmade with E-value of 0015 and 2500 sequence identityThe final structure was shown with RMSD value 12227A

Sequencing 7

P major MT2P ovata MT2

S lycopersicum MT2BN glutinosa MT2

A deliciosa MT2

C arabica MT1

A thaliana MT2A

B juncea MT22

B rapa pekinensis MT2B rapa MT2

A thaliana MT2B

B juncea MT25B juncea MT23

R communis MT2C arietinum MT2

T repens MT2V faba MT2

M domestica MT2

B juncea MT21

Hyd

ropa

thy

scor

e

16120804

0minus04

minus08

minus12

minus16

10 20 30 40 50 60 70 80

N-terminal cysteinerich domain

C-terminal cysteinerich domain

C-terminal cysteinerich domain

RMSD value 12227

N-terminal cysteinerich domain

a b c d e

a b

c

d

e

Spacer

Chain A

Template PDB ID 36G5C

119864-value 0015

Sequence identity 2500

Figure 5Modeling and analysis of PoMT2 Five distinct motifs of PoMT2 are indicated as a b c d and e and colored black on the 3DmodelHydropathy scores of each amino acid (position 5ndash77) of PoMT2 are shown at corresponding regions Multiple sequence alignment (shadingshowed residues identical for the given position in most sequences) showing relationship between PoMT2 and other plant MTs includingPlantago majorMT2 Solanum lycopersicumMT2B Nicotiana glutinosaMT2 Actinidia deliciosaMT2Malus domesticaMT2 Coffea arabicaMT1Arabidopsis thalianaMT2ABrassica junceaMT22Brassica rapa subsp pekinensisMT2Brassica rapaMT2Arabidopsis thalianaMT2BBrassica juncea MT21 Brassica juncea MT25 Brassica juncea MT23 Ricinus communis MT2 Cicer arietinum MT2 Trifolium repens MT2andVicia fabaMT2 (Uniprot IDs are Q5ZF75 Q40158 Q40396 P43390 O24058 P43396 P25860 P69163 Q39269 P69164 Q38805 P56168P56172 P56170 P30564 Q39459 P43398 and Q41657 resp)

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Biochemistry Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 6: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

6 Sequencing

6

5

4

3

2

1

0

Nor

mal

ized

relat

ive e

xpre

ssio

n

Control 20 120583M 40 120583M 80 120583MCuCl2 concentration in media

Figure 4 Real-time PCR expression profile of PoMT2 normalizedto the housekeeping gene actin under 0 20 40 and 80120583M copperstress The results indicate means of three replicates with theirstandard deviation

using iPSORT predicted that it does not have any mitochon-drial targeting peptide or chloroplast transit peptide UsingDiANNA server it was found that the PoMT2 has 7 predicteddisulfide bond between 3ndash8 4ndash75 10ndash67 14ndash20 16ndash78 23ndash80and 69ndash73 amino acid positions The terminal domain con-tained 0 aromatic 263 nonpolar aliphatic 605 polarand 105 charged amino acids In case of spacer region thesevalues were 93 419 302 and 186 respectively Thisprotein sequence of PoMT2 was compared using ClustalWand Blastp indicates that PoMT2 has 84 sequence identityto theMT2 encoded by Plantagomajor followed byAvicenniamarina 72 identity and Citrullus lanatus at 70 It hasbeen reported that class I MTs contain 20 highly conservedC residues showing homology to mammalian MTs whereasMTs without this strict arrangement of cysteines are referredto as class II MTs These MTs do not show homology tomammalian MTs Type 2 MTs contain CC CXC and CXXCmotifs in the N-terminal and CXC motif in the C-terminaldomain X can be any amino acid other than cysteine It alsocontains a CGGCmotif at the end of theN-terminal cysteine-rich domain and a spacer of approximately 40 amino acidresidues [12 15] In the present experiment PoMT2 followsall these criteria confirming that the isolated metallothioneinis a class II Type 2 plant metallothionein

The reverse transcription PCR product for ubiquitinwas sequenced A 204 bp sequence was obtained (Figure 2)(encoding 67 amino acids) which was searched for similarother sequences with the help of BLAST tool as describedabove It was found that the cloned sequence showed a goodsimilarity with some other published ubiquitin sequencesFor further confirmation the deduced amino acid sequencewas also searched by BLASTp tool and 99 sequenceidentity was found with other reported ubiquitin sequencesof Arabidopsis thaliana Plantago major Musa acuminateand Medicago truncatula (GenBank accession numbers areAAB95251 CAH56488 AAQ07454 and AET01627 resp)Finally it was submitted to GenBank with the accession

number JQ419758 It was the first ubiquitin sequence of Povata

32 Expression of PoMT2 Transcript in Different Stress Condi-tion To characterize PoMT2 transcript accumulation totalRNA extracted from various stress conditions were subjectedto reverse transcription (RT)-PCR reaction The seedlingswere treated with 0 20 40 and 80 120583M of CuCl

2solutions

for 24 h and 72 h (Figure 3) In each case 25 cycles of PCRreaction with first strand cDNA was carried out All thereactions showed the presence of a 214 bp bandThe intensityof the band was maximum in 40M CuCl

2treated sample

(after 24 h) which was 425 higher as compared to controlPoMT2 expression level was upregulated by all the threeconcentrations of CuCl

2after 72 hThe expression of PoMT2

was found to be higher in 40 120583M (1446) and 80120583M (111)treated samples than control In our experiment we havecompared the expression level of MT2 with that of ubiquitinunder similar experimental conditions The result indicatedan enhancement of MT type 2 expression with increasingdoses of copper treatment

Real-time PCR was used for more precise analysis ofexpression pattern of metallothionein type 2 gene Theresults of real-time PCR clearly indicated stress inducedaccumulation of MT2 transcript (Figure 4) At lower dosesthe expression of MT2 slightly increased with respect tocontrol but at higher doses a marked increase in expressionhas been observed Highest expression (49-fold) was foundin 40120583M treated sample However in 80 120583M treated sampleMT2 expression was decreased slightly than 40 120583M dose butstill remained higher (42-fold) than control These resultsare clearly in agreement with the data obtained in reversetranscription PCR Almost similar expression pattern wasreported for MT2 in Arabidopsis [18] In this species MT2amRNA was induced strongly whereas MT2b mRNA levelsincreased only slightly upon exposure to copper [29] Inanother experiment Physcomitrella patens MT2 transcriptwas also induced by copper stress [30] Significant increasein the MT2 transcripts level of Bruguiera gymnorrhiza wasalso found in response to heavy metals like zinc copperand lead [31] The current data suggest that higher doses ofcopper stress upregulate MT2 expression in plant which inturn results in a protection system against this metal

33 Structure Prediction and Phylogenetic Analysis Homol-ogy modeling was used to construct PoMT2 structureThis protein has three significant regions The N-terminal(residues 1ndash23) and C-terminal (residues 67ndash81) Cys richmetal binding domains were fairly conserved with othersequences whereas the spacer region (residues 24ndash66) wasvariable These three regions of PoMT2 were modeledtogether using ModWeb server As the protein data bankdid not contain any plant MT structure with a sequencethat would be sufficiently similar to the complete sequenceof this protein human ADAM22 (Protein Data Bank ID3G5C) was used as template structure 3D structure wasmade with E-value of 0015 and 2500 sequence identityThe final structure was shown with RMSD value 12227A

Sequencing 7

P major MT2P ovata MT2

S lycopersicum MT2BN glutinosa MT2

A deliciosa MT2

C arabica MT1

A thaliana MT2A

B juncea MT22

B rapa pekinensis MT2B rapa MT2

A thaliana MT2B

B juncea MT25B juncea MT23

R communis MT2C arietinum MT2

T repens MT2V faba MT2

M domestica MT2

B juncea MT21

Hyd

ropa

thy

scor

e

16120804

0minus04

minus08

minus12

minus16

10 20 30 40 50 60 70 80

N-terminal cysteinerich domain

C-terminal cysteinerich domain

C-terminal cysteinerich domain

RMSD value 12227

N-terminal cysteinerich domain

a b c d e

a b

c

d

e

Spacer

Chain A

Template PDB ID 36G5C

119864-value 0015

Sequence identity 2500

Figure 5Modeling and analysis of PoMT2 Five distinct motifs of PoMT2 are indicated as a b c d and e and colored black on the 3DmodelHydropathy scores of each amino acid (position 5ndash77) of PoMT2 are shown at corresponding regions Multiple sequence alignment (shadingshowed residues identical for the given position in most sequences) showing relationship between PoMT2 and other plant MTs includingPlantago majorMT2 Solanum lycopersicumMT2B Nicotiana glutinosaMT2 Actinidia deliciosaMT2Malus domesticaMT2 Coffea arabicaMT1Arabidopsis thalianaMT2ABrassica junceaMT22Brassica rapa subsp pekinensisMT2Brassica rapaMT2Arabidopsis thalianaMT2BBrassica juncea MT21 Brassica juncea MT25 Brassica juncea MT23 Ricinus communis MT2 Cicer arietinum MT2 Trifolium repens MT2andVicia fabaMT2 (Uniprot IDs are Q5ZF75 Q40158 Q40396 P43390 O24058 P43396 P25860 P69163 Q39269 P69164 Q38805 P56168P56172 P56170 P30564 Q39459 P43398 and Q41657 resp)

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Biochemistry Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 7: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

Sequencing 7

P major MT2P ovata MT2

S lycopersicum MT2BN glutinosa MT2

A deliciosa MT2

C arabica MT1

A thaliana MT2A

B juncea MT22

B rapa pekinensis MT2B rapa MT2

A thaliana MT2B

B juncea MT25B juncea MT23

R communis MT2C arietinum MT2

T repens MT2V faba MT2

M domestica MT2

B juncea MT21

Hyd

ropa

thy

scor

e

16120804

0minus04

minus08

minus12

minus16

10 20 30 40 50 60 70 80

N-terminal cysteinerich domain

C-terminal cysteinerich domain

C-terminal cysteinerich domain

RMSD value 12227

N-terminal cysteinerich domain

a b c d e

a b

c

d

e

Spacer

Chain A

Template PDB ID 36G5C

119864-value 0015

Sequence identity 2500

Figure 5Modeling and analysis of PoMT2 Five distinct motifs of PoMT2 are indicated as a b c d and e and colored black on the 3DmodelHydropathy scores of each amino acid (position 5ndash77) of PoMT2 are shown at corresponding regions Multiple sequence alignment (shadingshowed residues identical for the given position in most sequences) showing relationship between PoMT2 and other plant MTs includingPlantago majorMT2 Solanum lycopersicumMT2B Nicotiana glutinosaMT2 Actinidia deliciosaMT2Malus domesticaMT2 Coffea arabicaMT1Arabidopsis thalianaMT2ABrassica junceaMT22Brassica rapa subsp pekinensisMT2Brassica rapaMT2Arabidopsis thalianaMT2BBrassica juncea MT21 Brassica juncea MT25 Brassica juncea MT23 Ricinus communis MT2 Cicer arietinum MT2 Trifolium repens MT2andVicia fabaMT2 (Uniprot IDs are Q5ZF75 Q40158 Q40396 P43390 O24058 P43396 P25860 P69163 Q39269 P69164 Q38805 P56168P56172 P56170 P30564 Q39459 P43398 and Q41657 resp)

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Biochemistry Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 8: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

8 Sequencing

10001000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

1000

995

675

184

910

78

102

268

551

747

736

806

818

677

400877

410

5119890 + 001

P major MT2(Q5ZF75)

P ovata MT2

S lycopersicum MT2B(Q40158)

N glutinosa MT2(Q40396)

A deliciosa MT2(P43390)

C arabica MT1(P43396)

A thaliana MT2A(P25860)

B juncea MT22(P69163)

B rapa pekinensis MT2(Q39269)

B rapa MT2(P69164)

A thaliana MT2B(Q38805)

B juncea MT25(P56172)

B juncea MT23(P56170)

R communis MT2(P30564)

C arietinum MT2(Q39459)

T repens MT2(P43398)

V faba MT2(Q41657)

M domestica MT2(O24058)

B juncea MT21(P56168)

Figure 6 Phylogenetic tree of PoMT2 with other uniqueMTs selected fromUniProtSwiss-Prot knowledgebaseThis figure showed only theclade where PoMT2 belong with an arrow indicating P ovataMT2The phylogenetic tree depicts interrelationship of different MT2 proteinscollected from different species shown as operational taxonomic units The Uniprot IDs for each protein is given in parenthesis with thepercentage bootstrap values along the branch length

with respect to the template The resulting structures of N-and C-terminal Cys rich domain showed a single 120572-helixand 120573-sheet respectively whereas spacer region showed five120573-sheets (465 of spacer) at the core of the protein Asimilar structure was found in the spacer region of Triticumdurum [15] According to the hydropathy plot (Figure 5) ofPoMT2 most of the amino acids residing in spacer regionshowed high score for hydrophobicity whereas the score wasoverall low forC-terminal amino acid residues Leucine (at 46position) showed the highest score of +1522 whereas Lysine(at position 68) showed the lowest score of minus1522

A phylogenetic tree (see Supplementary Figure inSupplementary Material available online at httpdxdoiorg1011552013756983) was constructed using Phylip 369package enabling 100 bootstraps Only the important (for Povata MT2) cluster of total phylogenetic tree is shown inFigure 6 The phylogenetic analysis suggested that PoMT2

was highly similar toMT2 of P majorThese twoMT2smightoriginate from a common evolutionary ancestry These twoproteins mainly differ with respect to the sequence of aminoacids in the spacer region The closest neighbor of P ovataMT2 happened to beMT2 of Solanum lycopersicum Multiplesequence alignment (Figure 5) of PoMT2 with other plantMT2s supported this data It revealed that both the terminalmetal binding regions were highly conserved in most ofthe species and the spacer regions varied between speciesThe hydropathy plot indicated that the spacer formed thecore scaffold of the proteins Basically the lack of sequenceidentity of this spacer region reflected different structuralmotifs for the same protein in different plants whereas theN- and C-terminal domains are conserved indicating theirfunctional importance Having different conformations butsimilar functionality which is apparent from our phyloge-netic analysis (Figure 6) would indicate that the same class of

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Biochemistry Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 9: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

Sequencing 9

proteinwould behave differentially under different stress con-ditions which would be guided by their 3D-conformationsIn PoMT2 at least five motifs could be identified which aredistinct in terms of the nature of the amino acids and wouldlikely contribute to a novel fold of the proteinThis behavioraldifference was reported in Polulus alba where it showed thatMT2 transcript level was decreased with increasing time ofcopper treatment [32]

4 Conclusion

A class II type 2 MT gene designated as PoMT2 wassuccessfully isolated from Plantago ovata The sequence ofthis protein showed similarity with other reported MT2sMolecularmodeling and characterization of this protein werecarried out The distinct amino acid sequences of P ovatabelonged to the spacer region Like other plantMT2s PoMT2was found to be upregulated under copper stress as a defenseof the plant systemThese data suggest that the spacer regionis not involved in heavy metal detoxification but it has anevolutionary significance Since detailed knowledge of theprotein structure is required to know its biological functionsthe predicted three-dimensional structures of PoMT2 willcertainly smooth the way to understand the guidelines forfuture experiments

Acknowledgment

The authors express sincere gratitude to University GrantsCommission (UGC) for the award of Research Fellowship inScience for Meritorious Students (RFSMSs) fellowship to thefirst author

References

[1] M Galisteo M Sanchez R Vera et al ldquoA diet supplementedwith husks of Plantago ovata reduces the development ofendothelial dysfunction hypertension and obesity by affectingadiponectin and TNF-120572 in Zucker ratsrdquo Journal of Nutritionvol 135 no 10 pp 2399ndash2404 2005

[2] J W Anderson L D Allgood J Turner P R Oeltgenand B P Daggy ldquoEffects of psyllium on glucose and serumlipid responses in men with type 2 diabetes and hypercholes-terolemiardquoAmerican Journal of Clinical Nutrition vol 70 no 4pp 466ndash473 1999

[3] A L Romero K L West T Zern and M L FernandezldquoThe seeds from Plantago ovata lower plasma lipids by alteringhepatic and bile acid metabolism in guinea pigsrdquo Journal ofNutrition vol 132 no 6 pp 1194ndash1198 2002

[4] J M A Hannan L Ali J Khaleque M Akhter P R Flatt andY H A Abdel-Wahab ldquoAqueous extracts of husks of Plantagoovata reduce hyperglycaemia in type 1 and type 2 diabetes byinhibition of intestinal glucose absorptionrdquo British Journal ofNutrition vol 96 no 1 pp 131ndash137 2006

[5] L Lombardi and L Sebastiani ldquoCopper toxicity in Prunuscerasifera growth and antioxidant enzymes responses of in vitrogrown plantsrdquo Plant Science vol 168 no 3 pp 797ndash802 2005

[6] H Panou-Filotheou A M Bosabalidis and S KarataglisldquoEffects of copper toxicity on leaves of oregano (Origanum

vulgare subsp hirtum)rdquoAnnals of Botany vol 88 no 2 pp 207ndash214 2001

[7] I Yruela J J Pueyo P J Alonso and R Picorel ldquoPhotoinhi-bition of photosystem II from higher plants effect of copperinhibitionrdquo Journal of Biological Chemistry vol 271 no 44 pp27408ndash27415 1996

[8] E Ptsikk EM Aro and E Tyystjarvi ldquoIncrease in the quantumyield of photoinhibition contributes to copper toxicity in vivordquoPlant Physiology vol 117 pp 619ndash627 1998

[9] T S Babu J B Marder S Tripuranthakam D G Dixon and BMGreenberg ldquoSynergistic effects of a photooxidized polycyclicaromatic hydrocarbon and copper on photosynthesis and plantgrowth evidence that in vivo formation of reactive oxygenspecies is a mechanism of copper toxicityrdquo EnvironmentalToxicology and Chemistry vol 20 no 6 pp 1351ndash1358 2001

[10] A R Sheldon and N W Menzies ldquoThe effect of copper toxicityon the growth and root morphology of Rhodes grass (Chlorisgayana Knuth) in resin buffered solution culturerdquo Plant andSoil vol 278 no 1-2 pp 341ndash349 2005

[11] S Clemens ldquoMolecular mechanisms of plant metal toleranceand homeostasisrdquo Planta vol 212 pp 475ndash486 2011

[12] C Cobbett and P Goldsbrough ldquoPhytochelatins andmetalloth-ioneins roles in heavy metal detoxification and homeostasisrdquoAnnual Review of Plant Biology vol 53 pp 159ndash182 2002

[13] N A L M Van Hoof V H Hassinen H W J Hakvoort etal ldquoEnhanced copper tolerance in Silene vulgaris (Moench)Garcke populations from copper mines is associated withincreased transcript levels of a 2b-type metallothionein generdquoPlant Physiology vol 126 no 4 pp 1519ndash1526 2001

[14] L Lanfranco A Bolchi E C Ros S Ottonello and P BonfanteldquoDifferential expression of a metallothionein gene during thepresymbiotic versus the symbiotic phase of an arbuscularmycorrhizal fungusrdquo Plant Physiology vol 130 no 1 pp 58ndash672002

[15] K Bilecen U H Ozturk A D Duru et al ldquoTriticum durummetallothionein isolation of the gene and structural character-ization of the protein using solution scattering and molecularmodelingrdquo Journal of Biological Chemistry vol 280 no 14 pp13701ndash13711 2005

[16] G Mir J Domenech G Huguet et al ldquoA plant type 2metallothionein (MT) from cork tissue responds to oxidativestressrdquo Journal of Experimental Botany vol 55 no 408 pp2483ndash2493 2004

[17] M Das and S Sen Raychaudhuri ldquoEnhanced development ofsomatic embryos of Plantago ovata forsk By additivesrdquo In VitroCellular and Developmental Biology vol 37 no 5 pp 568ndash5712001

[18] A Murphy and L Taiz ldquoComparison of metallothionein geneexpression and nonprotein thiols in ten Arabidopsis ecotypesCorrelation with copper tolerancerdquo Plant Physiology vol 109no 3 pp 945ndash954 1995

[19] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[20] M T Araya A Siah D Mateo et al ldquoSelection and evaluationof housekeeping genes for haemocytes of soft-shell clams(Mya arenaria) challenged with Vibrio splendidusrdquo Journal ofInvertebrate Pathology vol 99 no 3 pp 326ndash331 2008

[21] S Castiglione C Franchin T Fossati G Lingua P Torrigianiand S Biondi ldquoHigh zinc concentrations reduce rooting capac-ity and alter metallothionein gene expression in white poplar

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Biochemistry Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 10: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

10 Sequencing

(Populus alba L cv Villafranca)rdquo Chemosphere vol 67 no 6pp 1117ndash1126 2007

[22] S -L The J O Abdullah and P Namasivayam ldquoMolecularcloning sequencing and characterization of a putative acetyl-CoA-C-acetyltransferase cDNA from a highly fragrant orchidhybrid Vanda Mimi Palmerrdquo Sequencing vol 2012 Article ID509034 8 pages 2012

[23] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmiR390-guided transacting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[24] P-A Binz and J H R Kagi ldquoMetallothionein molecularevolution and classificationrdquo inMetallothionein IV C KlaassenEd pp 7ndash13 Birkhauser Basel Switzerland 1999

[25] I El Ghazi B L Martin and I M Armitage ldquoMetallothionein-3 is a component of amultiprotein complex in themouse brainrdquoExperimental Biology and Medicine vol 231 no 9 pp 1500ndash1506 2006

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] U Pieper B M Webb D T Barkan et al ldquoMODBASE adatabase of annotated comparative protein structure modelsand associated resourcesrdquo Nucleic Acids Research vol 39 pp465ndash474 2011

[28] N Eswar B John N Mirkovic et al ldquoTools for compara-tive protein structure modeling and analysisrdquo Nucleic AcidsResearch vol 31 no 13 pp 3375ndash3380 2003

[29] J Zhou and P B Goldsbrough ldquoStructure organization andexpression of the metallothionein gene family in ArabidopsisrdquoMolecular and General Genetics vol 248 no 3 pp 318ndash3281995

[30] S H Cho Q T Hoang Y Y Kim et al ldquoProteome analysis ofgametophores identified a metallothionein involved in variousabiotic stress responses in Physcomitrella patensrdquo Plant CellReports vol 25 no 5 pp 475ndash488 2006

[31] G-YHuang andY-SWang ldquoExpression analysis of type 2met-allothionein gene inmangrove species (Bruguiera gymnorrhiza)under heavy metal stressrdquo Chemosphere vol 77 no 7 pp 1026ndash1029 2009

[32] A Macovei L Ventura M Dona M Fae A Balestrazzi andD Carbonera ldquoEffect of heavy metal treatments on metalloth-ionein expression profiles in white poplar (Populus alba L) cellsuspension culturesrdquoAnaleleUniversitatii DinOradea-FasciculaBiologie vol 27 no 2 pp 274ndash279 2010

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Biochemistry Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Page 11: Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein …downloads.hindawi.com/archive/2013/756983.pdf · 2019-07-31 · sequences of metallothionein type 2

Submit your manuscripts athttpwwwhindawicom

Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Advances in

Virolog y

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioinformaticsAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2013

ISRN Microbiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PeptidesInternational Journal of

ISRN Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Stem CellsInternational

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

ISRN Cell Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Biochemistry Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

ISRN Molecular Biology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

Evolutionary Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013


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