苏云金芽胞杆菌营养期杀虫蛋白Vip3Aa杀虫特异性机制研究

Due to its remarkable toxicity to lepidopteran Noctuidae pests, and no cross resistance with Cry1A proteins which have been commercialized successfully for the first generation transgenic crops, Vip3Aa protein from Bacillus thuringiensis has been widely used in transgenic maize and cotton. The binding between Vip3Aa and the receptors on insect midgut is crucial to exert virulence. Whereas, the investigation of insecticidal specificity mechanisms has not been performed well.. Among many vip genes cloned in our team, it was found that toxicity of vip3Aa39 ismuch higher than that of vip3Aa11 against Agrotis ypsilon, one of the most important agricultural insect pests, but toxicity of vip3Aa11 is much higher than that of vip3Aa39 against Helicoverpa armigera. Interestingly, both of them are high activity against Spodoptera exigua. There are 39 amino acid differences between this two kinds of protein. In this project, we will compare the binding difference between Vip3Aa11 and Vip3Aa39 with A. ypsilon and H. armigera as target insects; reveal the key receptor of insecticidal specificity through ligand fishing platform, 2D-DIGE and mass spectrometry technologies. Furthermore, we will detect crucial amino acid residue sites of Vip3Aa resulting in binding difference and pesticidal spetrum change by mutation.. This study will lay a solid foundation for revealing the mechanism of insecticidal specificity, and provide theoretical guidance and material conditions to broaden insecticidal spectrum and improve toxicity via directed modification.

苏云金芽胞杆菌Vip3Aa蛋白因其对鳞翅目夜蛾科害虫杀虫效果显著，与第一代商业化应用的Cry1A蛋白无交互抗性，目前已广泛用于抗虫转基因玉米和棉花。Vip3Aa与昆虫中肠受体结合是发挥毒力的关键，目前对其杀虫特异性机制研究尚未深入。. 在本项目组克隆的众多vip基因中，发现vip3Aa39对重要的农业害虫小地老虎毒杀活性比vip3Aa11高，对棉铃虫活性却比后者低，但对甜菜夜蛾均具高活性；这两种蛋白有39个氨基酸差别。本研究拟以小地老虎和棉铃虫为对象，通过配体垂钓平台、2D-DIGE及质谱等技术比较Vip3Aa11和Vip3Aa39蛋白对小地老虎和棉铃虫结合差异，发现杀虫特异性关键受体；拟通过构建突变体，进一步确定引起结合差异而导致杀虫谱变化的Vip3Aa蛋白关键氨基酸位点。本研究将为揭示杀虫特异性机制奠定坚实基础，为Vip3Aa蛋白拓宽杀虫谱、提高毒力等定向改造提供理论指导和物质条件。

苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt）营养期杀虫蛋白Vip3A类对夜蛾科害虫具有较高的杀虫活性。虽然Vip3Aa蛋白间的序列相似性很高（95%~100%），但是，氨基酸序列相近的Vip3Aa蛋白的杀虫谱和杀虫活性存在很大差异，本研究基于Vip3Aa11和Vip3Aa39蛋白氨基酸序列同源性高而杀虫活性差异大的特点，运用突变的方法对Vip3Aa11杀虫活性关键氨基酸进行了探讨，筛选出活性改变的突变子，为改造Vip蛋白奠定了理论及实验基础。.利用突变技术，明确了19个杀虫活性相关氨基酸位点和3个与杀虫特异性相关的氨基酸位点。筛选出对甜菜夜蛾（Spodoptera exigua）杀虫活性提高的突变子7个（S193T、S726T、F760L、E761G、S543N、I544L和S686R），杀虫活性降低的1个（N633T）。对棉铃虫（Helicoverpa armigera）杀虫活性提高的突变子4个（S9N、S193T、S194L、D641A），杀虫活性降低的9个（R115H、I358V、I362M、K553I、I663T、S543N、I544L和S686R、N624A）。Ser543 、Ile544、Ser686在Vip3Aa11杀虫特异性中可能发挥着关键作用。这一结果将为今后Vip蛋白的改造及结构与功能的研究奠定了良好的基础。.Vip3Aa11与 Vip3Aa39在棉铃虫中肠BBMV上存在竞争结合。Vip3Aa11蛋白在棉铃虫中肠BBMV上结合的受体蛋白大小为120 kDa、100 kDa和70 kDa。.这项研究不仅为 Vip3Aa 蛋白杀虫机制的研究拓宽思路，同时也为其它 Vip3Aa 蛋白扩大杀虫谱和提高杀虫活性的定向改造提供理论指导。