AGRICULTURAL RESEARCH COMMUNICATION CENTRE

www.arccjournals.comLegume Res., 37 (2) : 133 - 138, 2014

IN-SILICO VALIDATION AND COMPARATIVE ANALYSIS OF CANDIDATE GENEENCODING PROLINE RICH PROTEIN IN LENS CULINARIS

Jitender Singh* , Juhi Bhardwaj, Pankaj Kumar, Priya Tomar, Anchal Rani,Rosy Rani, Amit Kumar Singh and Anil Sirohi

College of BiotechnologyS.V.P. University of Agric. and Technology, Modipuram-250 110, India

Received: 21-09-2012 Accepted: 14-05-2013

ABSTRACTIn present study a proline rich protein has been identified in lentil EST database using (soyabean)

glycine max proline rich protein sequence as query sequence known to be down regulated in responseto salinity and drought stress. Sequence similarity search has shown 90 % similarity with chickpeaEST sequence Accesion no. GR 398344 and 85% similarity with Lens culinaris EST sequence Accessionno. GT 622346. Primer pairs were designed for this sequence and validated on selective Lens culinariscultivars. tblastx result has also shown similarity with the glycine max (Soybean) proline richprotein showing repetitive pattern of amino acids . Repetitive pattern observed was similar to thecharacteristic pattern present in proline rich protein. Identification of this gene will further help tostudy the role of proline rich protein in response to various biotic and abiotic stress.

Key words :- EST, Lentil, Proline rich protein.

*Corresponding author’s email: jeets_80@rediffmail.com

INTRODUCTIONDuring the past decade, several genes that

are regulated by biotic and abiotic stress have beenidentified (Tabaeizadeh, 1998). Proline rich protein(PRP) are cell wall proteins known to be influencedby factors associated with biotic and abiotic stresses,suggesting that the synthesis of the proteins issensitive to external stimuli (Tierney et al.,1988;Sheng et al.,1991; Marcus et al., 1991). Theseproteins play an important role in normaldevelopment, conferring the integrity of plant cell walland the structure maintenance of organs. Theseproteins have their own unique distribution patternsamong the various organs, tissues, and cell of plants,playing an integral role in the extracellular matrixstructure of many plant cells (Vasner and Lin 1989).So far most of the studies regarding proline richprotein is done with regard to plant development(Ye et al, 1991).

In the present study a new proline richprotein is identified in lentil EST database usingsoyabean proline rich protein sequence as querysequence. Lentil (Lens culinaris L.) is a diploid

(2n= 2x= 14), autogamous species and is one of theoldest crops in the world, which originated in theNear East (Zohary, 1972). Lentil is a plant that canadapt itself to arid and semi-arid climate fromdrought stress towards the end of the growing seasonin rainfed Medi terranean farming systems.Identification of this gene could help in selection ofsuperior drought tolerant lines in lentil cultivars andto study the role of proline rich protein gene inresponse to various biotic and abiotic stress.

MATERIALS AND METHODSIdentification of Prp2 gene in Lentil ESTDatabase and analysis through tblastn search:EST Sequences of PRP2 gene (NM001251664) fromGlycine max was retrived fron Genbank (http://w w w . n c b i . n l m . n i h . g o v / g e n b a n k /GenbankSearch.html). This sequence was used asquery against chickpea EST database and Lensculinaris. EST database in tblastn search (http://www.ncbi.nlm.nih.gov/BLAST.html). Sequencesshowing maximum similarity were downloaded fromgenbank (http://www.ncbi.nlm.nih.gov/genbank/GenbankSearch.html) and clustering was done using

DOI: 10.5958/j.0976-0571.37.2.020

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500bp < --------

B.A.FIG 1: PCR based amplification of proline rich region in Lens culinaris;A. M: Marker (1kb ladder, Fermentas), 1-6 Amplified PCR product with Multiple BandsB. M: Marker (1kb ladder, Merk Bioscience), 1 Negative control, 2-4 Amplification of ~ 230 bp from Lens culinaris

bioedit tool (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Expasy tool RADAR (Rapid AutomaticDetection and Allignment of Repeats) (http://www.ebi.ac.uk/Tools/Radar.html) was used toidentify repeat motifs in the Amino acid sequenceof prp2 gene.

Primer designing and validation of Prp2 gene:Primer was designed using Gen fischer tool (http://bibiserv.techfak.uni-bielefeld.de/genefisher2/submission.html) using optimum parameters forprimer designing like GC Content (40-60%). Tm(55°C), Product length (250bp), etc. Total DNA fromLens culinaris was isolated from fresh plant materialusing the plant genomic DNA Extraction Kit (QiagengmbH, Hilden, Germany) according to themanufacturer’s instructions. The DNA was storedin TE at -20 oC. Amplification was performed witha thermocycler (Biometra, T Personal 48) in 50 µlreaction containg 1X of PCR buffer, 2mM MgCl2,200 µM of each dNTP (MBI, Fermentas), 100mMPF/PR primers and 1U of Dream Taq (MBI,Fermentas). Each cycle consisted of denaturation at94°C (30 s), primer annealing at 58°C (30 s), andextension at 72°C (45 s), and a final extension of 10min at 72°C. The amplified DNA was excised andeluted from the gel using QlAquick gel extraction kit(Qiagen). The purified PCR product was ligated intopTZ57/T cloning vector (Fermentas). CompetentEscherichia coli (strain DH 5a) were transformedby standard molecular biology procedures(Sambrook and Russell 2001). Recombinant cloneswere identified by colony PCR or restrictionendonuclease digestion and selected clones were

sequenced at the automated DNA sequencingfacility, BioServe Biotechnologies (India) Pvt LtdBioserve, Hyderabad and the sequence so obtainedwas confirmed for the presence of PRP2 gene bydoing blastn and tblastn (http://www.ncbi.nlm.nih.gov/BLAST.html). Sequence wasalso matched with expasy tool Interproscan. Thesequence was submitted to GenBank with accessionnumber: JX564851.

RESULTS AND DISCUSSIONBLAST n and tBLASTn results: Approximately250 bp amplicons were obtained in Lens culinaris(Fig.1). Sequences were aligned using CLUSTAL Wand comparison of aligned sequences was done withBioEdit Sequence Alignment Editor (Fig. 2) (Hall,1999). Blast n results of the lentil sequence haveshown 85 % similarity with soyabean (Glycine max)proline rich protein (NM001251664) and chickpeaEST sequence (GR 398344). Pisum sativum(AJ233399), Gossypium hirusitum (AF044205) andPhaseolus vulgaris (AM158278) have also shownconsiderable similarity. There were 9 conservedrepeats of 18 amino acid each in case of glycineand chickpea wi th the repeat sequence“KPPVYKPPTEKPPVYKPP”. In case of lentilconserved repeats were present wi th“ V E N P P A Y K L P V E K H A V Y KPPVEKPPVYKPPVEKPPVYKPPVEKPPVYKPPVVKPPVYKPPVGYQPPVYTPPI “amino acideach was having repetitive sequences (Fig.3).Interproscan results have also shown similarity withthe proline rich protein (IPR003883) in PfamDatabase (Fig.4).

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FIG.2: Nucleotide Sequence alignment of Lens culinaris, Glycine Max, Medicago truncatula, Phaseolus vulgaris, C. arietiumand Pisum sativum sequences using Bioedit tool.

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FIG 4: InterproScan Results for proline rich protein in Lens cullineris

FIG. 3: Amino acid based sequence alignment of Proline rich region in Lens culinaris, Glycine Max, Medicago truncatula,Phaseolus vulgaris, C. arietium and Pisum sativum sequences using Bioedit tool.

PRPs are cell wall proteins in plant (Chenand Varner 1985; Tierney et al., 1988). Theseproteins can be classified into five groups on thebasis of motifs, domains and biochemical characters(Wang et al., 2006). The first group is characterizedby PRPs which contain tandem copies of thepentapeptide PPVXK/T (X is often H, Y or E) asappeared in MtPRP2, SbPRP2, (Wilson and Cooper1994; Hong et al., 1987). The second group is

characterized by two domain proteins, whichcontain a Proline-rich N-terminal domain withtandem repeats of PPYV motifs, and a C-terminaldomain that lacks Proline-rich sequences, such asthe sequence of AtPRP1/3 (Fowler et al., 1999). Incase third group of PRPs Cys rich domain is presentat C terminal end as it is present in AtPRP2 andAtPRP4. PEPK repetitive motifs are present in fourthgroup of PRP proteins. The fifth group of PRP

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characterized by the tandem repeats of PKPE, P(V/E)PPK in the C terminal of the protein sequence, asfound in OsPRP1.1-4 (Wang et al. 2006).

The prp2 gene identified in lentil has shownsimilarity with Glycine max prp2 gene which comesunder first group having multiple copies of PPVXK.Plant PRPs are expressed in response to manyexternal factors. Harrak et al (2001) had identifieda gene encoding a Pro-, Thr-, and Gly-rich protein(PTGRP) in case of Lycopersicon chilense whichwas negatively regulated by drought. Down-regulation of the PTGRP gene was also observed indesiccated cell suspensions of Lycopersicon chilense(Harrak et al., 2001). There is also a report onMsPRP2, a salt inducible gene encoding a PRPisolated from alfalfa (Deutch and Winicov, 1995).AtPRP3 in Arabidopsis was detected exclusively inroot (Fowler et al., 1999). Medicago truncatulaproline-rich protein (MtPPRD1) act as an lipid transferprotein (LTP) in membrane biogenesis and regulationof the intracellular fatty acid pool by binding andtransferring fatty acids and phospholipids between

membranes. It was also found that it controls thedevelopmental process specific to late germinationand to early phases of post-germination (Bouton etal., 2005) In case of Rice flower specific proline-richprotein in rice (OsPRP3) enhances the cell wallintegrity in the cold tolerant plant and confers cold-tolerance (Gothandam et al., 2010). SoyabeanProline-rich Protein (SbPRP) gene was identified insoybean known to be down regulated in response tosalt and drought stress (He et al., 2001).

Further, study of expression patterns inresponse to various biotic and abiotic stresses willprovide various clues to understand the functionalrole of PRP2 gene in lentil. Precise study ofsubcellular location and transgenic study will providea good elucidation of its biological functions.

ACKNOWLEDGEMENTSThis work was supported by Centre of

Excellence in Agriculture Biotechnology, Council ofScience & Technology, Uttar Pradesh andBioinformatics Infrastructure Facility funded by DBT,Government of India.

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