苏云金芽胞杆菌Vip3Aa11蛋白C端点突变对其杀虫活性的影响

The Influence of C-terminus Site-directed Mutation on the Toxicity of Bacillus thuringiensis Vip3Aa11 Protein

为研究Vip3Aa11羧基端对其杀虫活性和敏感性的影响,本研究利用定点突变技术构建了Vip3Aa11的3个突变体S543N、D547E和T681V。经SDS-PAGE分析证实3个突变体蛋白均能在大肠杆菌中表达分子量约88 k D的目的蛋白,生物活性测定显示,与Vip3Aa11相比,突变体S543N对甜菜夜蛾Helicoverpa armigera的杀虫活性提高了5倍。突变体D547E对甜菜夜蛾杀虫活性显著降低。突变体S543N、D547E和T681V对棉铃虫Spodoptera exigua的杀虫活性无明显变化。说明Vip3Aa11 C端部分氨基酸的定点突变对其杀虫活性有影响,且对不同害虫的杀虫活性变化趋势不同。本研究比较了Vip3Aa11蛋白与突变蛋白之间杀虫活性的差异,为研究Vip3Aa类蛋白的结构和机理奠定基础。

Vegetat ive 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 Vip3Aa protein der ived from different Bt strains are highly different,although the similarit ies between these proteins are higher than 95%.To determine whether the residues from the Vip3Aa11C-terminus contribute to the insecticidal activity,three mutants（S543 N,D547 E and T681 V） of Vip3Aa11 were constructed by site directed mutagenesis.Through SDS-PAGE,the mutants produced a 88 kD fusion protein,indicating that all mutants had successful expression in E.coli.Bioassay detection indicated that the insecticidal activity of the S543 N mutant against Spodoptera exigua increased obviously,and the LC50 value was approximately 5-fold higher than that of the Vip3Aa11,while the activity of D547 E mutant against S.exigua decreased obviously.The activities of all the mutants against Helicoverpa armigera had no obvious changes.The results indicated that the site directed mutagenesis of some amino acids from Vip3Aa11C terminus had an effect on insecticidal activity,and the effect was not consistent against different Lepidopteran pests even in the same mutant.In this study,the differences of insecticidal activity between Vip3Aa11 and mutant proteins serve a guideline for the study of Vip3Aa protein structure and activity mechanism.