棉铃虫钙粘蛋白胞内和胞外结构域在Bt毒素Cry1Ac毒杀过程中的作用

Bt cotton has been intensively used in China for several years. Resistance evolved by the cotton bollworm (Helicoverpa armigera) will directely affect the life span of Bt cotton in China. Cadherin is an important receptor of Bt toxin Cry1Ac in H. armigera, and mutated cadhrin alleles were genetically linked with high levels of resistance to Cry1Ac.Large-scale planting of Bt cotton created strong selection pressure on the target insect pest H. armigera,and increased frequecy of mutated cadherin alleles was detected in some field populations from northern China cotton area.However, how the mutated cadherin produces resistance to Cry1Ac is far from clear in H. armgiera. In this proposed project, we will target on two special resistance alleles of cadherin isolated from field populations of H. armigera in order to clarify relative contributions of cytodomain and ectodomain of cadherin in mode of action of Cry1Ac. One allele (r-LEV) has three resistance-related amino acid substitutions, and the other allele (r-cyto) has a 55 aa deletion in the cytoplasmic domain of cadherin. Based on the two resistance alleles, several fusion genes and genes with point mutations were constructed with overlap PCR and site-directed mutagenesis techniques. These wild-type and mutated cadherin alleles will be expressed in Sf9 cell lines, and their expression products will be functionally examined through a set of experiments (such as ligand blotting,cytotoxicity et al.). Our project is designed to elucidate the effects of cadherin alleles mutated at different positions or with different types on cytotoxicity of Cry1Ac with the Sf9 cell expression system. The expected results from this research will not only improve our understanding of mode of action of Bt toxin Cry1Ac, but also helpful to predict potential resistance-conferring mutations in cadherin and to investigate the rule of resistance evolution in field populations of H. armigera.

棉铃虫钙粘蛋白是Cry1Ac的重要功能受体之一，已证明钙粘蛋白基因突变与棉铃虫Cry1Ac高水平抗性遗传连锁。但是，钙粘蛋白突变导致棉铃虫Cry1Ac抗性产生的机理尚未明确。目前所发现的Bt抗性相关的昆虫钙粘蛋白基因均为胞外区氨基酸缺失或提前终止翻译，尚未发现与Bt抗性相关的胞内区突变或胞外区氨基酸点突变。本项目将利用从棉铃虫田间种群筛选获得到的2个独特的钙粘蛋白抗性等位基因(r-cyto:胞内区缺失55个氨基酸；r-LEV:胞外区氨基酸替换），通过重叠PCR和定点突变技术构建融合基因和点突变基因，采用Sf9细胞进行功能表达，并进行毒素受体结合、细胞毒力等测试，以明确钙粘蛋白突变部位（胞内、胞外）和突变方式（缺失、氨基酸替换）与Cry1Ac抗性的关系。本项目将阐明棉铃虫钙粘蛋白胞内区和胞外区在Bt毒性中的相对贡献，丰富Bt毒素作用机制的理论，并为预测棉铃虫钙粘蛋白抗性相关突变提供理论依据。

棉铃虫是一种重要的全球性害虫。目前，已在包括棉铃虫在内的多用鳞翅目昆虫中发现中肠钙粘蛋白突变可以对Bt毒素Cry1Ac产生抗性。在已发现的基于钙粘蛋白突变的抗性等位基因中，绝大部分是在钙粘蛋白胞外区发生改变，并导致高水平的隐性抗性；而在棉铃虫钙粘蛋白r15等位基因中，胞内区缺失了55个氨基酸，对Cry1Ac的抗性为不完全显性。为了明确棉铃虫钙粘蛋白胞内区和胞外区在Cry1Ac作用机理及抗性机理中的作用，本项目共构建了8种钙粘蛋白胞内和胞外突变体，在Sf9细胞中进行功能表达，并研究了Cry1Ac结合能力和细胞毒力高低与钙粘蛋白突变类型的关系。表达野生型和缺失胞外区CR1-CR9突变基因的Sf9细胞既能结合Cry1Ac又对Cry1Ac敏感；表达胞外结构域突变基因的细胞中，缺失毒素结合区（TBR）的突变体均不能结合Cry1Ac同时对Cry1Ac不敏感；而表达缺失胞内区的Sf9细胞尽管能与Cry1Ac结合，但是对Cry1Ac的敏感性显著低于表达野生型钙粘蛋白的细胞。上述结果表明，棉铃虫钙粘蛋白胞内区和胞外区均参与了Cry1Ac的毒杀作用过程。本项目在国际上首次明确了钙粘蛋白胞内区在Cry1Ac作用机理中的作用，丰富了对Bt作用机理的认识，对于靶标害虫基于钙粘蛋白突变的Bt抗性的监测、预警具有重要意义。