苹果蠹蛾代谢高效氯氟氰菊酯GSTs基因的鉴定及互作机制研究

谷胱甘肽转移酶（GSTs）是重要的解毒酶系，参与昆虫对杀虫剂的代谢作用，其酶活性升高是苹果蠹蛾对高效氯氟氰菊酯产生代谢抗性的主要机制。申请人前期从苹果蠹蛾基因组中鉴定出29个GSTs基因，并发现Delta家族2个GSTs基因可以代谢高效氯氟氰菊酯。是否还有其它能直接代谢高效氯氟氰菊酯的GSTs基因，以及代谢过程中这些GSTs与高效氯氟氰菊酯的互作机制尚不清楚。本项目拟利用荧光定量PCR分析GSTs基因的时空表达模式和杀虫剂胁迫下的表达模式；利用RNAi和生物测定明确干扰候选GSTs基因表达对杀虫剂的敏感性变化；结合定点突变、异源表达、体外代谢、分子模拟和计算丙氨酸扫描技术解析GSTs基因对杀虫剂的代谢功能，揭示代谢过程中GSTs与杀虫剂的互作机制。该项目的实施有助于阐明苹果蠹蛾GSTs代谢拟除虫菊酯杀虫剂的分子机制，同时也为构建以GSTs为防治靶标的苹果蠹蛾RNAi防控技术提供科学依据。

Glutathione S-transferases (GSTs) are important detoxification enzymes that play roles in metabolism of insecticides. Increased glutathione S-transferases (GSTs) activity was found to be the major metabolic resistance mechanism in the codling moth Cydia pomonella resistance to lambda-cyhalothrin. In the priminary study, we identified 29 GST genes from the genome database, and found that recombinant CpGSTd1 and CpGSTd3 enzymes were able to metabolize the mosr commonly used insecticide lambda-cyhalothrin. However, whether there are other GSTs genes could directely metabolize lambda-cyhalothrin and the interaction mechanisms between GSTs and lambda-cyhalothrin in the metabolism process are less known. In this research, RT-qPCR will be used to analysis the spatiotemporal and expression patterns of GSTs under lambda-cyhalothrin stress. RNAi and bioassay will be performed to determine the effects of interference of the expression of candidate GSTs genes on the changes of sensitivity of larvae to lambda-cyhalothrin. Combination of site-directed mutagenesis, heterologous expression, in vitro metabolism, molecular simulation, as well as computational alanine scanning techniques, the metabolic function of GSTs against lambda-cyhalothrin will be analyzed, and the interaction mechanisms between GSTs and lambda-cyhalothrin in the metabolism process will be revealed. The results of this study will help to illuminate GSTs mediated molecular mechanism in metabolism of pyrethroid insecticides, and provide scienticif basis for construction of RNAi-based C. pomonella control strategy with GSTs as target.