柑桔全爪螨P450基因的多样性及其介导代谢抗性的分子机理研究

The increased metabolic capacity of cytochrome P450 enzymes is one of the major reasons for insect and mite developed resistance to pesticides. This project intends to clarify the key scientific question about the diversity and mediated molecular mechanisms of metabolic resistance of P450 genes, and target the citrus red mite (Panonychus citri) as research object. On the basis of our pre-investigations on the resistance selection, biochemical and toxicological assay, transcriptome and digital expression profiles sequencing and analyses of citrus red mite, by using comparative transcriptomics, gene cloning, microarray, heterologous expression, cell bioassay, RNAi and chromatographic analysis, the gene annotation, ontology, and expression level analysis of P450 genes from transcriptome of different strains of citrus red mite will be analyzed. The P450 genes related to acaricide metabolism will be identified and the full-length cDNA sequences will be cloned, and the metabolism pathway as well as their encoded protein interactions will be analyzed. The molecular mechanism of 5-8 key P450 genes-mediated metabolic resistance in citrus red mite will be systematically revealed by analyzing acaricide metabolism of the recombinant proteins and the gene function validation through RNAi. Finally, the molecular diagnostic techniques for field population resistance will be developed with real-time quantitative PCR (RT-qPCR) technologies. The results will reveal P450-mediated metabolic resistance mechanism in theory and guide the integrated control of resistance in citrus red mite in practice.

细胞色素P450酶代谢能力增强是虫螨抗药性产生的主要原因之一。本项目针对虫螨P450基因的多样性及其介导药剂代谢抗性的机理这一科学问题，以柑桔全爪螨为研究对象，在项目组前期有关该螨抗药性筛选、生化毒理学分析、转录组及数字表达谱测序分析的基础上，综合运用比较转录组学、基因克隆、微阵列、异源表达、细胞生测、RNAi及色谱分析等技术和方法，通过对敏感和抗性品系转录组中P450基因的功能注释、富集及转录水平分析，筛选鉴定与药剂代谢相关的P450基因，克隆差异表达P450基因的cDNA全长序列并分析代谢通路和蛋白互作网络；系统解析5-8个重要基因异源表达重组蛋白对药剂的代谢特性及沉默后的功能，阐明P450基因介导该螨代谢抗性的分子机理，建立基于RT-qPCR技术的田间种群抗性分子诊断技术。研究结果将在理论上揭示害螨P450介导代谢抗性的分子机理，在实践上对害螨抗性治理及新药剂研发具有重要的指导意义。

本项目针对虫螨P450基因的多样性及其介导药剂代谢抗性的机理这一科学问题，以柑桔全爪螨为研究对象，在项目组前期有关该螨抗药性筛选、生化毒理学分析、转录组及数字表达谱测序分析的基础上，综合运用比较转录组学、基因克隆、微阵列、异源表达、细胞生测、RNAi及色谱分析等技术和方法，通过对敏感和抗性品系转录组中P450基因的功能注释、富集及转录水平分析，筛选鉴定与药剂代谢相关的P450基因，克隆差异表达P450基因的cDNA全长序列并分析代谢通路和蛋白互作网络；系统解析5-8个重要基因异源表达重组蛋白对药剂的代谢特性及沉默后的功能，阐明P450基因介导该螨代谢抗性的分子机理，建立基于RT-qPCR技术的田间种群抗性分子诊断技术。研究结果将在理论上揭示害螨P450介导代谢抗性的分子机理，在实践上对害螨抗性治理及新药剂研发具有重要的指导意义。