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University of Agricultural Sciences, Dharwad (2008)

Cloning of a drought inducible full length cDNA PjPlat1 from Prosopis juliflora

Mohammad Irfan.Inamdar

Titre : Cloning of a drought inducible full length cDNA PjPlat1 from Prosopis juliflora

Auteur : Mohammad Irfan.Inamdar

Université de soutenance : University of Agricultural Sciences, Dharwad

Grade : Master of Science (MS) in Plant Biotechnology 2008

Résumé
In the present study, drought inducible expression of transcripts coding for proteins with Cys-2/His-2 and PLAT domain was checked using RT-PCR with mRNA isolated from stressed and non-stressed plants of Prosopis juliflora. RT-PCR with Cys-2/ His-2 related degenerate primers produced 180 and 320 bp amplicons both from stressed and non-stressed mRNA, indicating that these transcripts were not differentially expressed. However, RT-PCR with PLAT-specific primers designed based on P. juliflora EST (DW359357) amplified a product (PjPLAT1) of 220 bp from stress induced mRNA only, but not from non-stress induced mRNA. Differentially expressed transcript was cloned in pTZ57R/T T/A cloning vector and sequenced. Upon BLASTn search PjPLAT1 showed homology to DW359567 EST of P. juliflora. Domain search indicated that the sequence contained PLAT domain-coding region, but it was partial. 5’and 3’ RACE-PCR gave a product of 600 and 350 bp, respectively. They were separately cloned into pTZ57R/T and sequenced. Contig assembly of RACE products gave an assembly of 921 bp. Primers were designed based on the assembled sequence, and employed for RT-PCR using stressed mRNA. Full length cDNA of PjPLAT1 (940 bp) contained 217 and 171 bp 5’ and 3’ untranslated region, respectively. Ribosome binding site (RBS) with a consensus motif of ATGG was found in the initiator codon. Also C (cytosine) was found both at -1 and -5 position as seen in the RBS of many eukaryotes. Poly A signal was observed from 834 to 839 bp. The full length PjPLAT1 cDNA had two ORFs ; ORF1 and ORF2. Deduced amino acid sequence of the two polypeptides consisted of 148 and 57 amino acids for ORF1 and 2, respectively. ORF1 showed a PLAT (Ploycysteine-1, Lipoxygenase, Alpha Toxin) domain (which is membrane associated and known to be drought inducible) homologous to that of Vitis vinifera (CAO67509), rice (NP001064434) and tomato (AAU03363

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