Cloning of a drought inducible full length cDNA PjPlat1 from Prosopis juliflora
Loading...
Files
Date
2008
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
UAS, Dharwad
Abstract
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).
Description
Keywords
Plant Biotechnology