小菜蛾田间种群抗性新基因GWAS定位及其功能研究

小菜蛾是为害十字花科蔬菜的主要抗性害虫，田间防控几乎陷入无药可用的被动局面，亟需解析其抗性机理以延缓杀虫剂抗性演化进程。目前，小菜蛾抗溴虫腈、甲维盐的分子机理尚未取得实质性进展，氯虫苯甲酰胺抗性除已报道因子外仍有未知因素。本项目拟利用前期采集的田间高抗种群构建F2家系群体，通过全基因组关联分析对三种新型药剂的抗性基因分别定位，采用图位克隆和序列筛查技术提取候选基因及突变信息。通过昆虫细胞-杆状病毒表达系统研究解毒酶类基因的体外代谢活性，利用爪蟾卵母细胞表达系统明确跨膜离子通道类基因的药剂响应效能，协同运用CRISPR/Cas9基因编辑系统阐明定位基因与抗性表型的因果关系。本项目将整合正向遗传学的经典定位策略和反向遗传学的最新技术手段，开展小菜蛾田间种群抗性新基因GWAS定位和功能研究，为开发小菜蛾对新型杀虫剂抗性检测方法和制订抗性治理对策提供科学依据，为基于靶标的新药研发提供重要筛选模型。

The diamondback moth, Plutella xylostella, is one of the major pest of cruciferous vegetables around the world. This pest has evolved resistance to nearly all kinds of insecticides, and fell into a passive situation that almost no efficient chemical for its control in the field. It is therefore necessary to make the resistance mechanisms clear, in order to delay the resistance evolvement. To date, however, the molecular basis of chlorfenapyr and emamectin benzoate resistance mechanisms remain unknown, and the novel factor involved in field-evolved chlorantraniliprole resistance still need to be investigated. In this project, we plan to mapping the resistance locus of the P. xylostella populations by using genome-wide association study approach. And then, the candidate genes will be identified through map-based cloning and sequence alignment technologies. Subsequently, Sf9 cell coupled with Baculovirus system will be employed to evaluate the detoxifying capable for insecticides of metabolizing enzymes, and using Xenopus oocytes system to examine the pharmacological properties of heterogenously expressed ion channels. Furthermore, CRISPR/Cas9-based functional studies will be performed to investigate the causal relationship between insecticide resistance phenotype and candidate gene. . In this study, we will integrate the classical forward genetics strategies and the latest reverse genetics approaches to identify the novel resistance genes in field population of P. xylostella. The expected results from this project will supply the scientific basis for develop molecular detection technology of resistance to new insecticides and design resistance management strategies, and provide important information on the discovery of target-based new chemicals.