苹果蠹蛾性信息素E8,E10-12:OH协同增效梨小食心虫性诱剂的分子机制

The oriental fruit moth, Grapholita molesta and the codling moth, Cydia pomonella, are closely related species in sympatric survival. Although the both female moths do not share same sex pheromone components, the major sex pheromone E8, E10-12:OH of codling moth has obvious synergistic effect on sex pheromone lures of G. molesta for trapping male moths. It was not clear that the maximum synergy and optimum composition of E8, E10-12:OH to sex lures. Hetero-specific pheromone components can synergize the effect that the male moths are more sensitive to its intranspecific sex pheromone lures, however, the molecular mechanism remains to be elucidated. In this study, we intend to obtain the optimum composition when E8,E10-12:OH showed maximum synergistic effect on sex pheromone lures of G. molesta via wind tunnel and trapping experiments. The olfactory receptor neurons responsible for recognizing the E8,E10-12:OH will be identified using Single-Unit Recording. The reason is going to be analyzed that OBPs of G. molesta can be “confused” by E8, E10-12:OH to transport it into the sensillum lymph by two phase binding assays, molecular simulation, and site-directed mutagenesis experiments. RNAi tests are applied to explore the recognition modes of ORs response E8,E10-12:OH and synergistic attractants. The results are theoretically creative to explain why the male insects can recognize the hetero-specific pheromone components, and also provide important references for developing synergistic sex lures of G. molesta with E8,E10-12:OH as an synergist.

梨小食心虫和苹果蠹蛾属同域近缘物种，两种食心虫并无共享的性信息素组分，但苹果蠹蛾性信息素E8,E10-12:OH对梨小食心虫性诱剂有显著的诱集增效作用。目前对E8,E10-12:OH增效性诱剂的最大能力及组分配比尚不清楚，其异源协同增效性诱剂的分子机制有待阐明。本项目拟通过风洞试验和诱捕试验确定E8,E10-12:OH具有最佳增效作用的组分配比；利用触角单感器记录测定识别E8,E10-12:OH的主要感受器神经元；通过双相结合试验、分子模拟和定点突变技术对异源性信息素E8,E10-12:OH“迷惑”梨小食心虫OBPs转运其进入感器的识别机理进行解析；利用RNAi试验对ORs响应E8,E10-12:OH及增效性诱剂混合气味分子的识别方式进行探究。研究结果对昆虫识别异源性信息素的理论有一定创新，对以E8,E10-12:OH为增效剂开发梨小食心虫高效性诱剂具有重要的参考价值。

本项目对E8,E10-12:OH具有最佳增效梨小食心虫性诱剂诱捕效果的组分配比进行了筛选，初步明确了梨小食心虫雄虫识别苹果蠹蛾性信息素E8,E10-12:OH的分子机制。田间诱捕结果表明，当Z8-12:Ac:E8-12:Ac:Z8-12:OH: E8,E10-12:OH = 100:6:2:20-40时（质量比），E8,E10-12:OH对梨小食心虫性诱剂具有最佳的增效效果，增效倍数达2.46倍。利用荧光竞争结合试验测定了梨小食心虫3种GmolPBPs和3种GmolGOBPs对E8,E10-12:OH的结合能力，发现GmolGOBP2对E8,E10-12:OH的结合能力显著高于其他5种GmolOBPs。分子柔性对接、定点突变和结合试验证实Glu104和Phe124是GmolGOBP2结合E8,E10-12:OH的关键残基。通过梨小食心虫触角三代全长转录组测序，基于基因注释、进化树分析等共鉴定到8种候选的性信息受体（命名为GmolOR1, 4, 6, 7, 8, 9, 11和15），qRT-PCR检测表明GmolOR1, 6, 7和8在雄虫触角中的表达量显著高于雌虫触角，GmolOR11在雌雄虫触角中的表达量均最高，推测以上5种GmolORs可能参与性信息素的识别。将以上5种GmolORs和共受体GmolOrco共表达于HEK293T细胞，荧光钙离子成像试验表明E8,E10-12:OH刺激表达GmolOR6、7和8的细胞系均能引起胞内钙离子浓度的剧烈变化，表明这些受体可以识别E8,E10-12:OH。通过RNA试验干扰敲低GmolOR1, 6, 7, 8和11基因的表达后，注射GmolOR6-dsRNA、GmolOR7-dsRNA和GmolOR8-dsRNA的雄虫对E8,E10-12:OH的EAG反应显著减弱，表明GmolOR6, 7, 8是识别E8,E10-12:OH的主要性信息素受体。