Research The Key Amino Acids Residues Of Insecticidal Activity From Bacillus Thuringiensis Vip3Aa

The vegetative insecticidal protein is a kind of new insecticidal protein secreted by Bacillus thuringiensis strains during vegetative growth,and has better insecticidal activity to Lepidoptera pests.The Vip proteins show no homologous to Cry protein and its insecticidal mechanism is different from Cry proteins.The Vip proteins can not only expand the Bt insecticide spectrum and provent or delay the production of agricultural pests resistance,it is very tempting application prospect,it also makes the Vip protein caused people’s attention increasingly.However,the conformational three-dimensional(3D)structure of Vip proteins has not yet been elucidated.The relationship between the structure and function has not been known,and some problems such as insecticidal activity needs further improve,so need to study the structure and function of the protein,looking for key amino acid of insecticidal activity.In this paper,the Vip3Aa11 insecticidal activity key amino acids are discussed by site directed mutagenesis,and established the random mutation method of Vip3Aa11,screening of active change mutation,which establishes the theoretical and experimental basis for the reconstruction of Vip protein.Although Vip3Aa protein sequence between difference less than 5%,but there is a big difference in insecticidal spectrum and insecticidal activity between different proteins of Vip3Aa.In this study,first of all,by making the amino acid sequence alignment of Vip3Aa39 and Vip3Aa11 which preserved in the laboratory,using the site-directed mutation technology,successfully constructed 9 mutation proteins in amino terminal and 19 mutation proteins in carboxyl terminal of Vip3Aa11.The bioassays used Helicoverpa armigera and Spodoptera exigua.In amino terminal,the bioassay results showed that insecticidal activity of the S9N and S193T mutants against Spodoptera exigua were increased 2-3 fold,the S9N,S193T and S194L mutants against Helicoverpa armigera were increased 1.5-3 fold.In carboxyl terminal,the bioassay results showed that insecticidal activity of the S726T,F760L and E761G mutants against Spodoptera exigua were increased 2.6-6 fold,the N470K,D611N,N691H and I706N mutants against Helicoverpa armigera were increased 1.5-3.8 fold.The R115H,1358V,I362M,K553I and 1663T mutants against Helicoverpa armigera were obviously depression.The SDS-PAGE results showed that,in addition to V1161 and T257A mutations,all variants can be expressed 88 kDa protein band at levels identical to those for wild-type Vip3Aa11.Both variants and wild-type proteins can yield a 62-66 kDa fragment by trypsin digestion in vitro,and the enzyme solution is consistent.We extrapolate differences in toxicity of variants which are not due to unstable protein.However,three-dimensional structure of Vip proteins has not yet been elucidated,the reasons for toxicity differences are unclear,and still need further design experiment to analysis,test and verify.The level of Vip3Aa protein expression in the wild Bt strain is low,and the expression of protein is not stable.Some research points out that the use of Bt Ⅰ-Bt Ⅱ promoter expression the vip3Aa gene encoding protein,can effectively improve the stability of the Vip3Aa protein and protein expression level.In this study,we have recombined crylAc promoter with vip3Aa11 gene by gene recombination technology,transformed into a host bacterium.The Vip3Aall protein expressed under the promoter cry1Ac and T7 in insecticidal effect,resistance to trypsin and fermentation condition were analyzed respectively,in order to improve the Vip3Aa protein expression,stability and even mprove its insecticidal activity.The results showed that the insecticidal activity of expressed protein under the promoter of crylAc and T7 has no significant difference to Spodoptera exigua and Helicoverpa Armigera respectively.In the amount of Vip3Aa11 protein expression,crylAc promoter was lower than T7 promoter.In the stability of antitrypsin,crylAc promoter is higher than T7 promoter.The results of this study would provide a new way for vip gene expression,function verification and insecticidal mechanism.At the same time,the results also provide new materials for random mutation of subsequent.The 3D structure of Vip3A protein has not been resolved,but,the previous study point out the mutation in carboxyl end of Vip3Aa11 can have a significant impact on its insecticidal activity.In this study,we studied the random mutation of Vip3Aa11 carboxyl end by error-prone PCR.Establish a random mutant library,which each mutant have 1.5 base mutation rates be expected,needs to be error-prone PCR conditions as follows:starting template amount 3.76 ng,PCR cycle number 15,using fluorescence quantitative PCR monitoring PCR amplification process.After a round of error-prone PCR,get 900 transformations.Randomly selected 30 transformants were analyzed for sequencing.The results showed that the mutant of nucleotide changes was 13,a total of 20 base point mutations,the average mutation 1.5 bases of each mutation.It is expectations.The biological activity of 13 mutant was assayed with Spodoptera exigua and Helicoverpa armigera.The results showed that two mutant(A10 and A21)was increased the insecticidal activity to Spodoptera exigua under the concentration of 2 ug/mL,and three mutant(A10,A20 and A21)was increased the insecticidal activity to Helicoverpa armigera under the concentration of 40 ug/mL.This method laid an important foundation for modify the protein and research the structure and function of Vip3 protein.