Functional characterization of novel RbTI gene from ricebean and validation of its insecticidal properties in transgenic tobacco.

Abstract

Plant protease inhibitors (PI's) inhibit the activity of gut proteases and thus provide resistance against insect attack. Previously we have published first report on cloning and characterization of a novel Bowman-Birk protease inhibitor gene (RbTI) from ricebean (Vigna umbellata). In this study, the RbTI gene was further characterized and validated as a potential candidate for transferring insect resistance in economically important crops. We have successfully generated transgenic tobacco plants expressing RbTI gene constitutively under CaMV35S promoter using Agrobacterium transformation. Genomic PCR and GUS analysis confirmed the successful integration of RbTI gene into tobacco plant genome. qRT-PCR analysis revealed highest RbTI gene expression in transformed tobacco leaves nearing maturity. Feeding of transformed tobacco leaf tissue showed prominent effect on larval mortality throughout the larval growth stages mainly during first three days of feeding. For functional analysis of RbTI gene, we estimated the inhibitory activity of protein extracts from normal and transformed tobacco plants against gut proteases of Spodoptera litura and H. armigera larval instars. Maximum inhibition of trypsin (82.42% and 73.25%) and chymotrypsin (69.50% and 60.64%) enzymes was recorded at early larval stages of both insects. The results of this study validated the future use of RbTI gene from ricebean legume as a potential candidate for transferring insect resistance in economically important crops. Insight, innovation, integration: Present study was conducted with the aim to utilize the state of art biotechnological techniques for transferring key pest resistant genes from underutilized promising crop ricebean. The tobacco plant has been utilized as modern plant for proof of concept where a protease inhibitor gene from Ricebean has been transferred to tobacco plant which induced larval mortality within first three days of feeding at all larval developmental stages. The biochemical assays on mid-gut total protein extract showed that the transgenic tobacco leaves have inhibiting effect on trypsin and chymotrypsin enzymes of insect which is otherwise required for digestion of food by them. Hence, we provide a novel gene that could be utilized for pest resistance in other crops different developmental stages.