棉铃虫基于异位不互补效应对Cry1Ac产生抗性的新基因鉴定及机制解析

Bt作物大规模种植取得了显著的经济效益和生态效益，但靶标害虫对Bt的抗性进化严重威胁Bt作物的可持续应用。在棉铃虫上已发现3种不同的Cry1Ac抗性基因：HaCad（钙粘蛋白）、HaABCC2（ABC转运蛋白）和HaTSPAN1（四跨膜蛋白）。棉铃虫LF256品系对Cry1Ac的抗性为隐性遗传且超过200倍，上述3种已知抗性基因均未产生突变且在遗传上与抗性不连锁。LF256品系的新型抗性基因与HaCad互作可以产生异位不互补效应。本项目拟通过全基因组关联分析（GWAS）对棉铃虫LF256品系Cry1Ac抗性新基因进行定位和克隆，用细胞表达和基因编辑分别对新基因进行离体和活体功能验证，并系统解析新基因和HaCad互作产生异位不互补效应的机制。该研究将发现一种新型的棉铃虫Cry1Ac抗性基因及其与HaCad互作产生抗性的机制，为开拓基因互作影响害虫Bt抗性进化这一新的研究领域提供重要理论基础。

Transgenic crops expressing insecticidal proteins from Bacillus thuringiensis (Bt crops) have been massively planted, and marked economic and ecological benefits have been achieved. However, evolution of resistance to Bt proteins by target pest insects is threatening the sustainable use of this green technology for pest control. Complexity in mode of action of Bt proteins has shaped genetic diversity of Bt resistance in target pest insects. Three different genes of Cry1Ac resistance, including HaCad (cadherin), HaABCC2 (ABC transporter) and HaTSPAN1 (tetraspanin), have been discovered in the cotton bollworm, Helicoverpa armigera. The LF256 strain of H. armigera has high level resistance to Cry1Ac (>200-fold), which is recessively inherited and not related to the three known resistance genes. However, the second-site noncomplementation (SSNC) was observed when crossing LF256 and the HaCad-mutant strain of H. armigera. The proposing project will map and clone the new Cry1Ac resistance gene in LF256 by using the genome-wide association study (GWAS). Then, functional validation of the resistance gene will be carried out with in vitro Sf9 cell expression system and in vivo genome editing technique. Finally, physiological mechanisms of resistance conferred by epistasis between the new resistance gene and HaCad will be dissected. The new project is expected to discover a new gene for Cry1Ac resistance and resolve the mechanisms of epistasis between the two resistance genes. This will help to strengthen our understanding of molecular basis of diversified resistance mechanisms to Cry1Ac in H. armigera. It will also provide theoretical basis for investigating impacts of epistasis on resistance evolution in target pest insects of Bt crops.