The Influence Of Fragment Replacement On The Toxicity Of Bacillus Thuringiensis Vip3Aa11 And Vip3Aa39

Bacillus thuringiensis(Bt)insecticide is currently the most effective and environmentally friendly microbial pesticide in the world.Vegetative insecticidal proteins(VIPs)are representative novel insecticidal proteins found after Insecticidal crystal proteins(ICPs)and have high insecticidal specificity against lepidopteran insects..The amino acid sequence of Vip3 Aa protein has high homology,but the insecticidal spectrum and insecticidal activity of Vip3 Aa protein derived from different Bt strains are different.Vip3Aa11 and Vip3Aa39 differ only by 39 amino acids,but exhibited different insecticidal activities against Lepidoptera pests such as Plutella xylostella,Spodoptera exigua,and Helicoverpa armigera.This article discussed the key amino acid fragments of Vip3Aa11 and Vip3Aa39 insecticidal activity,laid a theoretical and practical basis for the application of Vip protein in agricultural insecticidesIn this study,based on the amino acid sequence differences of Vip3 Aa protein,the amino acid fragments of Vip3Aa11 and Vip3Aa39 were replaced by overlapping PCR technology,and eight substitutional proteins were successfully obtained: 11N-39 C,39N-11 C,11N-11M-39 C,39N-39M-11 C,11N-39M-39 C,39N-11M-11 C,11N-39M-11 C,39N-11M-39 C.Then the replacement gene and the vip3Aa11 and vip3Aa39 genes were induced in E.coli BL21(DE3)strain.The SDS-PAGE pattern showed that the vip3Aa11,vip3Aa39 and the eight permutants were expressed in soluble fractions approximately 88 kDa protein fragments.Vip3Aa11,Vip3Aa39 protein and eight substitutional proteins were used to measure indoor biological activity of Plutella xyllostella,Spodoptera exigua and Helicoverpa armigera,respectively.Bioassay results showed that compared with Vip3Aa11 and Vip3Aa39 proteins,the insecticidal activity of the substitutional proteins to Plutella xylostella and Spodoptera exigua declined to varying degrees,and the insecticidal activity against Helicoverpa armigera did not change significantly.The 11N-39M-39 C,39N-39M-11 C,11N-39M-39 C and 39N-11M-11 C four-substance substitutions were screened for reduced insecticidal activity against Plutella xylostella,Spodoptera exigua,and Helicoverpa armigera;11N-39M-Both 11 C and 39N-11M-39 C had no significant changes in activity against Plutella xylostella,Spodoptera exigua,and Helicoverpa armigera;11N-39 C and 39N-11 C substitutions had reduced insecticidal activity against Plutella xylostella and Spodoptera exigua.However,there was no significant change in the activity of Helicoverpa armigera.The results of trypsin digestion showed that the Vip3Aa11 and Vip3Aa39 proteins could form a stable 62 kDa resistant core polypeptide after digestion,and the substituted 11N-39 C,39N-11 C,11N-39M-11 C and 39N-11M-39 C were also digested.Into a 62 kDa resistant core polypeptide,the substitutions 11N-11M-39 C,39N-39M-11 C,11N-39M-39 C,and 39N-11M-11 C were not digested as resistant core fragments.The results suggest that the first 210 amino acids at the N-terminus and the 524 to 789 amino acid fragment at the C-terminus of the Vip3Aa11 and Vip3Aa39 proteins are key fragments affecting the insecticidal activities of these three pests.This provides clues for studying the relationship between the structure and function of Vip3 A proteins.