The Influence Of C-terminus Site-directed Mutation On The Toxicity And Sensitivity Of Bacillus Thuringiensis Vip3Aa11 Protein

Vegetative insecticidal proteins(VIPs)of Bacillus thuringiensis show a wide insecticidal spectrum against a wide variety of lepidopteran pests.The insecticidal activity and specificity of the Vip3 Aa protein derived from different Bt strains were highly different,although the similarities between these proteins is higher than 95%.To determine whether the residues from the Vip3Aa11C-terminus contributed to the insecticidal activity and sensitivity.this study based on the high sequence homology and the differences in insecticidal activity between Vip3Aa11 and Vip3Aa39 proteins.nine different amino acid residues of Vip3Aa11 were substituted with the corresponding amino acid residues from Vip3Aa39 by site-directed mutagenesis.Nine mutations were obtained:S543N,I544 L,D547E,T681 V,685I,S686 R,R704G,I780 L and Y784 N.All genes were expressed in the transformed E.coli BL21 strain.Through SDS-PAGE,the expressed products included a fusion protein at a band of 88 kDa,indicating that all mutants were successfully expressed in E.coli.The bioassays used four Lepidoptera insects(Plutella xyllostella,Agrotis ipsilon,Helicoverpa armigera,and Spodoptera exigua).The bioassay results showed that all mutants and Vip3Aa11 protein lacked obvious insecticidal activity against Plutella xyllostella.Compared with Vip3Aa11,the insecticidal activity of mutant proteins to three Lepidoptera insects(Agrotis ipsilon,Helicoverpa armigera,and Spodoptera exigua)had different changes.The bioassay results showed that the insecticidal activity of the S543 N,I544L,and S686 R mutants against Spodoptera exigua were obviously increased,and the insecticidal activity were approximately 5-,2.65-,and 8.98-fold higher than those of the wild type,while their toxicities against Helicoverpa armigera and Agrotis ypsilon was decreased to some extent.The mutant Y784 N lost its insecticidal activity against four Lepidoptera species.The trypsin activation of the nine mutants indicated that mutant Y784 N was found to be highly sensitive to trypsin digestion,and it was completely degraded by trypsin,which might be the root cause of the loss of insecticidal activity.while the other eight active mutants generated a trypsin-resistant polypeptide of 62 kDa upon trypsin digestion.These finding indicate that some amino acids between 543-784 of the Vip3Aa11 C-terminus have important effects on the insecticidal specificity and insecticidal toxicity,and the amino acid sites required for different pests are also different.This study laid an important foundation for revealing the insecticidal activity and specificity of Vip3Aa11 protein.