Cloning, Characterization and Evaluation of Toxicity of Newly Identified Vip3Aa Proteins from Bacillus Thuringiensis Recovered from Diverse Environments for Biological Control of Helicoverpa Armigera

Ten full-length vip3-type genes amplified from the Bacillus thuringiensis (Bt) isolates recovered from various Indian agroclimatic zones and two from known Bt strains were cloned, sequenced and confirmed as vip3Aa-type genes based on sequence homology. Comparison of deduced amino acid sequences of these genes compared with previously reported vip3Aa-type genes and other lepidopteran-specific vip3Aa genes revealed new amino acid substitutions, indicating the diversity of Indian Bt isolates. These genes have been grouped into six phylogenetic clusters and assigned names as vip3Aa67-vip3Aa78 by Bt nomenclature committee (www.bpprc.com). Interestingly, the substitution of lysine in the holotype Vip3Aa1 with glutamine at amino acid position 284 was uniformly found in all our Vip3Aa-type proteins, suggesting sequence conservation as well as diversity among these proteins. For evaluation of toxicity, vip3Aa67-vip3Aa72 genes were cloned in pET-29a(+) Escherichia coli expression vector. The immunostrip assays, SDS-PAGE and western blot analysis with Vip3A antibodies confirmed the presence of Vip3Aa67, Vip3Aa68, Vip3Aa69, Vip3Aa70, Vip3Aa71 and Vip3Aa72 proteins of ca. 89 kDa size. The toxicity of these Vip3Aa proteins along with the Vip3Aa44 protein used as a positive control was evaluated against Helicoverpa armigera. LC50 ranged from 0.921 to 8.513 ppm indicating their insecticidal potential. The impact of Vip3A proteins on the life cycle characteristics of H. armigera was studied, and the results showed toxic and sublethal effects on the growth and fecundity of the insect population. Computational analysis of the three-dimensional structure of Vip3Aa proteins revealed the effect of amino acid substitutions on stability, highlighting the importance of flexibility in protein function. This study reveals the potential of the newly identified Vip3Aa proteins in the biological control of H. armigera.