Cloning and characterization of protease inhibitor genes from some legumes

Abstract

Protease inhibitors are natural defense proteins that inhibit the proteolytic activity of proteases. Legume protease inhibitors are diverse in their molecular mechanism and many of them have successfully been deployed in crop plants to confer resistance against insect pests. Nine combinations of degenerated primers,designed, based on “multiple aligned” gene sequences from NCBI Gene bank database were used to amplify the protease inhibitor genes in of ten different legume crops (two varieties each)viz.fieldpea,chickpea, lentil, lathyrus, pigeonpea , frenchbean,mungbean ,clusterbean , horsegramand cowpea .The primer combination BBF2/BBR2 amplified the expectedamplicon of 800 bp invarities ofmungbean,horsegramand cowpea. These amplicons obtained from each genotype were further ligated to the pJET1.2/ blunt vector and transformed into XL1 blue strain of E.coli. Out of 41 clones sequenced, two clone sequences obtained from cowpea (DCS 6) showed homology with substilisin-like serine protease inhibitor of Desulfovibria vulgaris and serine/threonine protein kinase of Epichlocfastucae.These genes can be transformed into Agrobacterium based binary vectors and used for transformation of crops. Therefore these findings offer new genes for transgenic development against lepidopteron pests.