小菜蛾谷氨酸门控氯离子通道阿维菌素受体位点的定位研究

Avermectins are widely used as one of the most effective control measures in the globe fight against arthropod pests and parasitic nematodes. Glutamate-gated chloride channels (GluCl) are the major targets for avermectins. Previous studies identified several mutations associated with abamectin/ivermectin resistance. However, the receptor sites for abamectin on GluCls, the contribution to abamectin resistance of single mutation, and effects on fitness of the genomic-edited strains remain elusive. The diamondback moth, Plutella xylostella, is one of the most devastating insect pests of cruciferous vegetables around the world, and lots of field populations have evolved high levels of resistance to abamectin. In this study, we will make use of 3D model-driven mutagenesis to predict the allosteric effects resulted from potential receptor residues mutation occurring at Q253, S294, N298, P302, A309 and G315 of GluCl channel of P. xylostella. We will also employ the Xenopus oocytes expression system and electrophysiology to confirm the pharmacological response to abamectin of the candidate residues, which might be involves in receptor sites. Furthermore, in order to clear whether the above six mutations will results in abamectin-resistant phenotype and fitness cost in P. xylostella, the CRISPR/Cas9 mediated genomic-editing will be performed subsequently. This study will mapping the receptor sites for abamectin on PxGluCl channel, by synthetically analyzing the molecular docking, two-electrode voltage clamp recording and genomic-editing results obtains in molecular, cellular and individual levels, respectively. Results from this work could facilitate implementing effective resistance monitoring and management strategies in the diamondback moth and other arthropod pests, as well as provide target model for novel avermectins development.

阿维菌素类药剂在世界范围内广泛应用于节肢动物害虫和寄生性线虫的防治，其最重要的生理靶标是谷氨酸门控氯离子通道。目前，已报道该通道基因突变与阿维菌素类抗药性相关，但阿维菌素的受体位点和点突变的抗性贡献及其对种群适合度的影响尚不明确。本项目拟利用全球性为害十字花科蔬菜、对阿维菌素已产生高水平抗性的小菜蛾为试虫，利用模型驱动的对接分析，预测潜在受体位点（Q253、S294、N298、P302、A309和G315）突变介导的通道变构效应，通过卵母细胞表达和电生理实验明确目标位点的药理学特性，并采用CRISPR/Cas9技术在小菜蛾基因组中对候选位点进行编辑，测定其对阿维菌素毒力和种群适合度的影响。本项目将在分子、细胞和个体水平对阿维菌素靶向谷氨酸门控氯离子通道的受体位点进行模拟对接、功能验证和综合解析，研究结果将为小菜蛾等有害生物的抗性监测和治理提供科学依据，为阿维菌素类新型药剂研发奠定理论基础。

小菜蛾是世界范围内严重为害十字花科作物的经济害虫，其抗药性演化能力极强，已对101种活性成分产生了不同程度的抗性，包括杀虫性能优良的阿维菌素类杀虫剂。前期研究表明，小菜蛾谷氨酸门控氯离子通道（PxGluCl）基因A309V突变可导致低水平的抗性，但阿维菌素靶向PxGluCl通道的受体位点尚不清楚。本项目明确了PxGluCl基因的结构特征，鉴定出节肢动物GluCls受体抗性相关变异的hot regions位于exon 3 和exon 9结构域。通过同源建模和分子对接技术，预测了Q253、S294、N298、T314四个氨基酸很可能是与通道结合的极性互作位点。利用爪蟾卵母细胞表达系统和双电极电压钳记录技术，明确了exon 9的三个可变剪接体具备不同的药理学特性，且exon 9c对阿维菌素的响应能力最弱（EC50 = 1.07 μΜ），该区域参与阿维菌素的受体位点组成。与野生型PxGluCl通道相比，Q253A和P302S突变型受体对阿维菌素的敏感性下降了约5倍，是该药剂的次要受体位点；但T314A通道对阿维菌素的敏感性降低了约100倍，G315E通道基本丧失了对药剂响应能力（EC50 = 80.32 μΜ），这两个位点是阿维菌素的主要受体位点。建立了小菜蛾CRISPR/Cas9基因编辑系统，成功实现了小菜蛾PxRyR受体突变的定点敲入，但PxGluCl基因编辑遇到同源重组修复效率低的问题，在6720个注射卵中仅检测到1头携带G315E突变的个体；成功获得了分别携带Q253A、T294P、A309V、T314A和G315E突变的果蝇品系，生物测定显示T294P和A309V纯合突变体对阿对菌素有中低等水平的抗性，诊断剂量下存活率约10%。此外，本研究在田间种群鉴定到PxGluCl基因的S294P和Δ295-296缺失型新突变，开展了基于第三代单碱基编辑器的基因敲入和基于piggyBac转座系统的基因转入技术在PxGluCl基因修饰中的探索性应用。综上，本项目综合利用多项正向遗传学和反向遗传学策略和技术，明确了小菜蛾PxGluCl通道基因变异介导的阿维菌素结合效能差异，鉴定出该药剂的主要和次要受体位点。研究结果丰富了小菜蛾PxGluCl基因介导阿维菌素抗性演化的基础理论，为节肢动物抗药性机制探索提供了重要借鉴。