Isolation and characterization of water stress activated protein kinase gene from black pepper (Piper nigrum L.) var. Kalluvally.

Abstract

Water stress is identified as one of the main constraint for enhancing the productivity in black pepper. To survive stress, plants employ a complex set of distinct signaling pathways that trigger stress-specific tolerance or avoidance in the organism as a whole. An important biochemical mechanism for regulating such pathways is reversible protein phosphorylation which is mediated by protein kinases. Gaining an understanding of the mechanisms that regulate the expression of these genes and functional annotation of their transcripts will be necessary for the genetic improvement of plants cultivated in extreme environments. Genotypic variations for drought tolerance have been reported in black pepper and the variety Kalluvally is one of the drought tolerant genotype of black pepper (Thankamani, 2003). In the previous studies at the Centre, water stress specific cDNA library of Kalluvally was constructed by suppression subtractive hybridization (SSH) (Kushwah, 2008). Several protein kinase genes were found to be up-regulated during the study. The present investigation was undertaken to obtain full length coding sequence information on the partial clones of protein kinase genes available in the cDNA library by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The clones named PNK21 and PNK49 were selected from the library. The clones were revived on LB/ampicillin media and subjected to full length sequencing. Degenerate primers were designed based on protein kinase gene sequences of various crops, obtained from NCBI database to amplify the unknown ends of the transcripts by RT- PCR. Plants raised under greenhouse were screened for revival after water stress with different interval and frequency under open condition. The total RNA was isolated using TRI® reagent from stressed plants and subsequently cDNA was synthesized. The PCR amplification was carried out using degenerate primers designed for all three clones. Amplicons of size of ~600 bp for PNK21 clone and ~600 bp for PNK49 clone were obtained. Direct sequencing of PCR product of PNK21 clone was done. The sequence data obtained was merged and analysed using bioinformatics tools. Blastn analysis revealed ~50 per cent coverage with the cDNA sequences for protein kinase from database. So, further primers were designed to amplify the full length cDNA sequence by RNA ligase mediated-RACE. The 3’ end of the PNK21 cDNA was successfully amplified using RLM-RACE PCR with amplicon size of ~400 bp. The purified PCR product was ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin with X-gal and IPTG. After confirmation of the insert by colony PCR, the clones were sequenced. The finally enriched sequence was analysed using bioinformatics tools. Blastn and Blastx revealed maximum similarity with Ricinus communis APK1B protein kinase. The sequence indicated open reading frame and conserved domains for protein kinase and polyadenylation signal site TATAAA was found just upstream of the polyA tail at 3’ end when analysed with different Bioinformatics tools.