Bacillus thuringiensis Vip3Aa and Vip3Ad Chimeric Proteins Improve their Insecticidal Activity against Lepidopteran Insects

Vegetative insecticidal proteins (Vips) are potential alternatives for Bacillus thuringiensis endotoxins.Vip3Aa39 showed high toxicity against lepidopterans, whereas Vip3Ad displayed no toxicity.In this study, sequence alignment analysis indicated that Vip3Aa39 and Vip3Ad share 85% homology with one another.Amino acid fragment interchange between Vip3Aa39 and Vip3Ad was carried out by the overlapping extension PCR method, and 6 chimeric genes were obtained successfully.These chimeric genes were transformed and expressed in Escherichia coli strain BL21.SDS-PAGE analysis confirmed the expression of 88 kDa proteins.Insect bioassays were conducted with Helicoverpa armigera and Plutella xylostella, and after the N-terminal amino acid fragment (1-60) of Vip3Aa39 was replaced by that of Vip3Ad protein, the recombinant protein Vip3AdAa1 exhibited high toxicity against both insects.Vip3AdAa2 (amino acids 1-118 replaced by that of Vip3Ad) displayed no significant difference in toxicity compared with Vip3Aa39.Vip3AaAd1 showed no toxicity against H. armigera and P. xylostella.The chimeric proteins Vip3AaAdAa1, Vip3AaAdAa2 and Vip3AaAd2 displayed differences in insecticidal spectra against the two insects.This study also evaluated the hydrolysis of chimeric proteins with trypsin.Five chimeric proteins were cleaved into 62 kDa fragments, except for Vip3AaAd1.These new Bt proteins constructed by protein engineering can effectively enrich insecticidal protein resources.This study also provides a reference for in-depth understanding of the key activity areas of Vip3A insecticidal proteins.