Identification of Heat-Related ESTs in Moth Bean through Suppression Subtraction Hybridization

Abstract

Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.