棉铃虫基于HaCad的Bt抗性基因超显性的形成机理

Diverse mutations of the cadherin gene (HaCad) are associated with Cry1Ac resistance in Helicoverpa armigera. Previous work has demonstrated that the resistant strain SCD-r1, which is homozygous for a truncated allele of HaCad (r1), has 540-fold resistance to Cry1Ac, and the SCD-r15 strain, which is homozygous for a cytoplasmic domain mutant allele of HaCad (r15), exhibits 140-fold resistance to Cry1Ac. However, the F1 progeny from crosses between SCD-r1 and SCD-r15 obtained 910-fold resistance, which is higher than either of the resistant parents. To analyze this overdominance phenomenon, we will examine binding and interactions between Cry1Ac and BBMV proteins from H.armigera larval midguts of different HaCad genotypes. Sf9 cell lines will be used to express r1, r15, and co-express them, respectively. The Sf9 cells expressing HaCad proteins will be evaluated by immunolocalization, real-time monitoring of binding with Cry1Ac toxin, and cytotoxicity bioassays et al. Our project will be expected to elucidate the molecular and biochemical mechanisms of overdominance of Bt resistance alleles at the cadherin locus of H. armigera, and the expected results from this research will provide an important theoretical basis for predicting potential evolution of HaCad-based Bt resistance in field populations of cotton bollworm.

棉铃虫钙粘蛋白基因（HaCad）的多种突变可以导致棉铃虫对Cry1Ac产生抗性。前期的研究发现：基于HaCad胞外区突变基因r1的棉铃虫品系SCD-r1 (r1r1)对Cry1Ac具有540倍抗性，基于HaCad胞质区突变基因r15的棉铃虫品系SCD-r15 (r15r15)对Cry1Ac具有140倍抗性，而SCD-r1和SCD-r15的杂交F1代 (r1r15)对Cry1Ac具有910倍抗性，表现为超显性现象（即杂合子的抗性高于任一亲本）。本项目拟比较棉铃虫HaCad不同基因型品系中肠BBMV与Cry1Ac互作的差异，同时利用Sf9细胞对r1和r15等位基因分别进行单基因表达和两个基因共表达，通过免疫定位、Bt毒素-受体实时互作监测、细胞毒力测定等研究，以阐明HaCad不同抗性等位基因互作产生超显性的分子和生化机制，并为预测基于HaCad的棉铃虫Bt抗性演化潜力提供重要理论依据。

棉铃虫钙粘蛋白胞质区突变品系SCD-r15(r15r15)与钙粘蛋白胞外区突变品系SCD-r1(r1r1)的杂交后代对Cry1Ac产生高水平抗性，表现出超显性现象。本项目比较了不同钙粘蛋白基因型品系中肠BBMV与Bt毒素Cry1Ac的互作，发现杂合基因型品系Cry1Ac结合量显著少于野生品系和两个突变品系，表明不同钙粘蛋白互作能够显著降低毒素与受体的结合。利用杆状病毒表达系统对这两个突变基因进行了单基因表达和共表达，比较了表达不同钙粘蛋白突变基因的细胞与毒素的结合能力及其对毒素的敏感性。结果发现，表达r1的细胞不能与毒素结合，并且对毒素不敏感。表达r15的细胞与共表达r1r15的细胞均能与毒素结合，但相对于r15，共表达r1r15的细胞与Cry1Ac的结合能力显著下降，并且其对Cry1Ac的敏感性显著低于表达r15的细胞对Cry1Ac的敏感性。以上结果表明钙粘蛋白胞质区突变基因与胞外区突变基因的互作能够显著影响Cry1Ac与受体的结合，从而产生超显性现象。超显性机制的揭示对于预测棉铃虫田间种群Bt毒素抗性的演化具有重要意义。