Vip3Aa毒素在斜纹夜蛾中肠疑似作用受体验证及其结合机制分析

With the emergence of pest resistance to Bt Cry toxin, the discovery and utilization of Vip toxin is a very urgent task.However, there is so far no report worldside about the receptor of Vip3Aa toxin in target insects and the binding mechanism between the toxin and receptor.In the previous research,we found three putative receptors of Vip3Aa toxin in Spodoptera litura midgut based on the transcriptional profiling analysis and the pull-down analysis. The objective of this proposal is to find the real receptor in S. litura midgut for Vip3Aa toxin and to investigate the binding mechanism between the toxin and receptor. The S. litura mutant stains will be obtained by using the Crispr/Cas9 system to knock out these putative receptor genes respectively,and then the susceptibility of mutant larvae to Vip3Aa will be assessed by bioassays.The binding experiment between Vip3Aa and BBMV,prepared from S. litura mutant larvae with the weakened susceptibility to Vip3Aa,will be set up to finally identify the real receptor of Vip3Aa in S. litura midgut.On the basis of the above results,the different mutants of Vip3Aa will be constructed to explore the key binding fragment and site. The results of this study will not only contribute to better understanding of the insecticidal mechanism of Vip3Aa, and also be significant for improving the insecticidal virulence, preventing insect resistance to B. thuringiensis and breeding of transgenic plants with vip3Aa gene.

随着害虫对Bt Cry毒素抗性的不断出现，Vip毒素的发现和利用是当前十分迫切的课题。然而，关于Vip3Aa毒素在靶标昆虫中肠的作用受体以及毒素-受体的结合机制国内外迄今未见报道。本项目拟在前期研究发现斜纹夜蛾幼虫中肠3个Vip3Aa毒素疑似受体的基础上，采用Crispr/Cas9技术分别对它们进行敲除，获得敲除突变体纯合子品系，筛选出对Vip3Aa敏感性减弱的斜纹夜蛾突变株系，通过突变株系中肠BBMV总蛋白与Vip3Aa毒素蛋白的结合实验确定Vip3Aa在斜纹夜蛾中肠的真正作用受体；在此基础上，采用片段缺失和定点突变构建Vip3Aa的突变体，通过生物测定和结合试验分析Vip3Aa上的结合关键区段和位点，揭示两者间的结合机制。预期结果有助于深化对Vip3Aa毒素作用机制的认识，对于Bt毒力提高、抗性治理与转基因植物的生产实践具有重要意义。

虽然苏云金杆菌Vip3Aa毒素已被广泛应用于鳞翅目害虫的防治，但Vip3类毒素蛋白的作用机制还尚不明朗。本研究首先确定了Bt Vip3Aa毒素在斜纹夜蛾肠道内活化后的主要活性蛋白复合物成分，并对其组成、结构、生化性质进行了分析，证明具备蛋白N端~19 kDa片段及C端~66 kDa片段的四聚体蛋白是Vip3Aa在斜纹夜蛾中肠内的主要毒性核心。基于对Vip3Aa活化后不同蛋白复合物杀虫活性的分析，本研究成功筛选出Vip3Aa毒素杀虫活性复合物较之无活性成分在斜纹夜蛾中肠内的特异性结合蛋白。通过建立斜纹夜蛾CRISPR/Cas9基因敲除技术平台，成功敲除了斜纹夜蛾中肠内多个基因，获得多个缺失了与Vip3Aa毒素分子潜在互作蛋白的斜纹夜蛾纯合子品系。目前已完成3个疑似受体的验证，其余特定基因缺失的斜纹夜蛾品系对Vip3Aa及其它Bt毒素敏感性的分析仍在继续进行中。本项目的顺利执行为Vip3Aa毒素杀虫机制的研究提供了依据，为进一步建立完整的Vip3Aa杀虫机制模型奠定了基础。