嘴壶夜蛾识别性信息素的分子机制

At present, studies on moth sex pheromone recognition were focused on the type I pheromone. Only a few genes for type Ⅱ pheromone recognition were identified from three geometrids and lymantrids. Oraesia emarginata was found with type Ⅱ pheromone in our previous study, and 7 candidate pheromone receptors (OemiPRs) and 3 candidate pheromone binding proteins (OemiPBPs) were screened out from our previous antennal transcriptome analysis. In the study, full length of OemiPRs and OemiPBPs will be cloned. Their structures will be predicted and compared to PRs and PBPs for type I pheromone recognition. The evolution of PR and PBP between O. emarginata and other moths will be analyzed too. Expression level of OemiPRs and OemiPBPs will be tested and their location in the sensillum will be ascertained by in situ hybridization. OemiPBPs with biological activity will be expressed in a bacterial system and their functions will be tested with PBP binding assays. To found the ligand of OemiPRs, their expression level in the antennae will be knocked down by RNA interference method and the EAG response of these antennae to pheromone will be studied. Our results will not only make clear the molecular mechanism of sex pheromone recognition, and the functional evolution of OemiPRs in O. emarginata, but also describe the evolutionary relationship between type I and type Ⅱ sex pheromone recognition genes, and laid a theoretical foundation for the development of efficient sex pheromone lure of O. emarginata.

蛾类性信息素，目前集中研究I型性信息素的识别机制，只报道了3种尺蛾和毒蛾的Ⅱ型性信息素识别基因。我们前期研究发现，嘴壶夜蛾使用Ⅱ型性信息素，触角中表达7个候选性信息素受体（PRs）和3个候选性信息素结合蛋白（PBPs）。本项目将依据克隆到的PRs和PBPs全长，分析其结构，并将其与I型性信息素识别基因比较，讨论它们的进化关系；分析PRs和PBPs的表达水平，并用原位杂交技术对它们在感器中的分布进行定位；利用蛋白表达技术在体外表达具有生物活性的PBP蛋白并结合荧光竞争实验分析它们的识别配体；利用RNAi技术调低触角中PR的表达水平并利用EAG技术分析触角对性信息素的反应差异，从而明确PR的功能。本项目的成功实施不仅可以明确嘴壶夜蛾识别性信息素的分子机制和PR基因的功能进化，而且可以在一定程度上阐述Ⅱ型和I型性信息素识别机制的进化关系，也为开发高效的嘴壶夜蛾性诱剂奠定理论基础。

目前集中研究蛾类I型性信息素的识别机制，只报道了3种尺蛾和毒蛾的Ⅱ型性信息素识别基因。我们前期研究发现，嘴壶夜蛾使用Ⅱ型性信息素，触角中表达7个候选性信息素受体（PRs）和3个候选性信息素结合蛋白（PBPs）。本项目首次获得了嘴壶夜蛾4个PRs、3个PBPs和嗅觉受体共表达基因OemaORCO的CDS全长。OemaPBP1、OemaPBP2和OemaPBP3的前19、24和10个氨基酸分别为信号肽。OemaOR4、OemaOR29和OemaORco的氨基酸序列能够预测出7个跨膜结构域，OemaOR21和OemaOR26则不能。PBPs和已知夜蛾科I类性信息素基因相似率约为66%，而PR最高仅58.3%。PRs和PBPs主要在成虫触角中表达，OemaOR4在蛹触角、OemaORco和OemaPBP2在幼虫和蛹触角中也有较高表达。OemaOR29、OemaOR4、OemaPBP2和OemaPBP3在雄蛾触角中高表达，OemaOR26和OemaOR21在雌蛾触角中高表达，OemaORco和OemaPBP3在雌雄蛾触角中高表达。OemaOR29、OemaPBP1和OemaPBP3主要表达在长毛型感器中，其他基因则在短毛型感器和（或）部分长毛型感器中有表达。成熟OemaPBP1、OemaPBP2和OemaPBP3蛋白的分子量分别为16.2KD、16.3KD和16.4KD。OemaPBP1在低温诱导8h时体外表达量大，OemaPBP3则需高温诱导。OemaPBP1识别的配体为性信息素cis-9,10-epoxy-Z6-21:H，对性信息素cis-9,10-epoxy-Z3,Z6-21:H也有一定结合能力，OemaPBP3蛋白对两种性信息素、Z11-16:Oac和Z3-6:Oac均有一定结合能力。OemaOR4 RNAi降低触角对 cis-9,10-epoxy-Z3,Z6-21:H的EAG反应，OemaOR29 RNAi降低触角对 cis-9,10-epoxy-Z6-21:H的EAG反应，OemaOR21 RNAi和OemaOR26 RNAi并不影响触角对使用化合物的EAG反应。本项目资助下还完成了草地贪夜蛾、二化螟和亚洲玉米螟的部分研究。本项目的成功实施不仅明确了嘴壶夜蛾识别性信息素的分子机制，而且为阐述Ⅱ型和I型性信息素识别机制的进化提供了依据。