细胞色素P450 CYP6CW1等位基因突变介导的褐飞虱对吡虫啉抗药性的分子机制

The brown planthopper, Nilaparvata lugens (BPH), is a major rice pest throughout Asia. Resistance to neonicotinoid insecticides has becoming the major threat for planthopper control. Deciphering the detailed molecular basis of such resistance is critical to designing diagnostic tools to implement suitable resistance management strategies. Our previous study showed that allelic variation of CYP6CW1 gene which containing two key amino acid mutations may contribute to the imidacloprid resistance in a field collected population. This foundation aims to further explore the basic resistance mechanism mediated by the allelic variation of CYP6CW1 gene. A near-isogenic BPH strain resistant to imidacloprid will be established and utilized for evaluating the role of allelic variation of CYP6CW1 in the regulation mechanism of the resistance to imidacloprid; in vitro (protein expression and metabolism) and in vivo (transgenic expression in Drosophila melanogaster) functional characterization tools will be applied to demonstrate that if allelic variation in CYP6CW1 is the key molecular change that helps BPH to acquire high resistance to imidacloprid insecticide. Furthermore, site-directed mutagenesis coupled with in vivo functional characterization will be used to detect which single amino acid change responsible for high imidacloprid resistance compared to susceptible allele. The results of this study will allow the design of DNA-based diagnostic assay to detect and track imidacloprid resistance in field populations in China, and have great meanings for implementing of suitable resistance management strategies.

褐飞虱是水稻最重要的害虫之一，其对新烟碱类杀虫剂的抗性已成为褐飞虱防治的最大障碍。深入研究其分子抗性机理是建立快速有效的抗性分子检测方法的重要基础。前期申请者在田间采集到一个吡虫啉抗性种群，经过研究发现细胞色素P450CYP6CW1等位基因突变可能与褐飞虱对吡虫啉的抗药性有关。本项目拟在此基础上通过：1）构建抗吡虫啉近等基因系，分析CYP6CW1等位基因突变的表达水平与抗性水平的关系；2）运用体外蛋白表达及体外杀虫剂代谢的方法分析CYP6CW1突变体对吡虫啉的代谢能力；3）运用体内果蝇转基因技术分析CYP6CW1突变体及关键性的氨基酸突变在吡虫啉抗性中的功能。这些研究旨在揭示CYP6CW1等位基因突变介导褐飞虱对吡虫啉抗药性的分子机制。研究结果对设计基因检测技术来进行褐飞虱田间种群新烟碱类杀虫剂抗药性监测和追踪提供科学依据，对褐飞虱田间种群抗性治理和综合防治具有重要的指导意义。

褐飞虱是水稻最重要的害虫之一，其对新烟碱类杀虫剂的抗性已成为褐飞虱防治的最大障碍。深入研究其分子抗性机理是建立快速有效的抗性分子检测方法和抗性治理的重要基础。本项目在田间多个吡虫啉抗性种群中发现了细胞色素P450 CYP6CW1v2基因编码区存在高频率的(110I/371A/414F)的突变，而此突变的频率在室内敏感种群中不到10%。运用分子对接技术初步预测此突变可能与褐飞虱对吡虫啉的抗药性有关。构建了P450 CYP6CW1单点突变和混合突变果蝇转基因突变品系，证实了414F突变对吡虫啉抗药性的贡献最大，CYP6CW1(110I/371A/414F)突变品系对吡虫啉的抗药性是CYP6CW1 (110T/371V/414S)变异体的三倍左右；体外蛋白表达及体外杀虫剂代谢技术表明CYP6CW1(110I/371A/414F)突变体对吡虫啉的代谢能力显著强于CYP6CW1 (110T/371V/414S)变异体。进一步深入研究表明 (110I/371A/414F)突变在2018年-2021年田间种群中普遍存在，且突变在中等吡虫啉的选择压力下只在筛选代数初期呈现连续的上升趋势；对采用敏感种群构建的(110I/371A/414F)和 (110T/371V/414S)纯合品系进行回交试验和基因型检测，发现(110I/371A/414F)突变与吡虫啉抗性显性具有显著性相关性，证明了吡虫啉低抗性阶段或吡虫啉中等或低选择压下此突变组合起到重要的抗性作用，在抗性后期还可能和其它抗性机制联合起到作用。研究结果揭示了CYP6CW1等位基因突变介导的田间褐飞虱对吡虫啉抗药性的普遍分子机制。研究结果对设计基因检测技术来进行褐飞虱田间种群新烟碱类杀虫剂抗药性监测和追踪提供科学依据，对筛选抵抗此CYP6CW1基因突变的杀虫剂进行褐飞虱田间种群抗性治理和综合防治具有重要的指导意义。