Cloning of drought related genes in Vigna aconitifolia through modified differential display

Abstract

A total of 12 genotypes of moth bean were evaluated for desiccation tolerance at germination and seedling stage. Differential response was observed at 8% and 15% PEG on germination and seedling level respectively where Varieties RMO-435, RMM-12-Single, RMM12-Poly And CZM-105 Were foundtolerant. Certain protein were found to be upregulated with treatment in all the samples while some showed upregulation only in tolerant types through SDS PAGE analysis. Twenty four primer combinations of anchored poly T (3) and random primers (8) were used to amplify the transcripts and amplicons were resolved on agarose gel with ethidium bromide. This modified DDRT-PCR assay amplified a total of 95 bands with an average of 4.04 per primer combination. 43 gene fragments were found to be up-regulated whereas 34 were downregulated in response to drought as compared to the control. A total of 23 unigenes (4 contigs and 19 singletons), were derived by cluster assembly and sequence alignment of 29 ESTs Out of 29 ESTs, 4 EST (13.79%) was found to be novel to moth bean. Significant BLASTX similarity (<1E06) in the NCBI non-redundant (nr) database was detected for 25 ESTs (86.20%). Gene ontology unctional classification terms (BLASTX results and GO terms), were retrieved for all sequences and 23 sequences were annotated with 8 enzyme commission (EC) codes and were mapped to 11 different KGG pathways Our study lists mainly the transcripts that are differentially expressed under PEG induced desiccation stress, where 9 chaperones including HSP 70, HSP 101 and Dnaj like proteins were nfound.