中华蜜蜂对狄斯瓦螨清理行为中的嗅觉信息识别和传递机理

As Chinese native bee species, Apis cerana cerana has sensitive olfaction, especially occupying the hygienic behavior to clean out the minatory ectoparasitic mite - Varroa destructor, which has done great harm to the Western honeybee, Apis mellifera. In order to realize the molecular mechanism of the unique behavior of bees, here we will firstly clone the differential olfactory receptor (ORs) and odorant binding protein (OBPs) gene sequences from Chinese honey bees' antenna based on the transcriptome difference between the A. cerana cerana and A. mellifera, and the following analysis are the spatio-temporal expression profiles and antennal immunohistochemical co-localization analysis; Meanwhile the reactive volatile odors causing the bees' hygienic behavior to cleaning mites will be separated and identified by the method of Gas chromatography ? electroantennographic detection (GC-EAD) and Gas chromatography ? mass spectrometry (GC-MS) from chemical volatile substance of V. destructor; Then the identified reactive chemical odors of mites will be test the binding profiles to the recombinant proteins, ORs and OBPs in vitro, respectively; Finally the binding interaction between ORs and OBPs proteins will be demonstrated with the technique of Fluorescence resonance energy transfer (FRET). In a word, based on the investigation of hygienic behavior to V. destructor through the unique odors recognition and transmission involved in the sensitive olfactory system of Chinese honey bee, the project will be helpful of the interpretation of unique olfactory physiological behavior mechanism of Chinese honey bee, and provide the theory evidence for the absolutely solving the difficulty of harm of V. destructor in the apiculture.

中华蜜蜂Apis cerana cerana是中国的本土蜂种，其嗅觉灵敏，可以凭借其非常独特的清理行为，对西方蜜蜂的重大外寄生螨- - 狄斯瓦螨Varroa destructor进行有效地清理以避免被其为害。为深入了解中蜂这种独特行为内在的嗅觉信息识别和传递分子机制，本研究拟首先根据中蜂和意蜂内勤工蜂触角转录组的差异分析结果，获得中蜂特异的气味结合蛋白OBPs和嗅觉受体ORs等嗅觉相关基因，并进行时空表达和触角免疫共定位分析；再对引起中蜂清理行为的瓦螨气味信息物质进行GC-EAD和GC-MS分析并结合嗅觉行为学测试；然后将中蜂OBPs与ORs分别与瓦螨气味物质进行嗅觉信息识别功能分析；最后利用FRET技术研究清理行为中的嗅觉信息传递功能。总之本项目将通过中蜂基于嗅觉系统分子识别传递的独特瓦螨清理行为机理研究，为解析中蜂独特的嗅觉生理行为学机理和彻底解决养蜂业中狄斯瓦螨为害提供理论依据。

中华蜜蜂Apis cerana cerana是我国的本土蜂种，其嗅觉灵敏，对意大利蜜蜂的重大敌害——狄斯瓦螨Varroa destructor，具有敏锐准确的识别和清理能力。为了深入了解中蜂这一独特行为内在的嗅觉信息识别和传递分子机制，本研究根据前期中蜂和意蜂工蜂触角转录组的差异分析结果，克隆获得了中蜂特异性的气味结合蛋白OBPs和嗅觉受体Ors等嗅觉相关基因，并对其进行时空表达和触角免疫共定位分析；同时对引起中蜂清理行为的瓦螨气味信息物质进行分析鉴定；最后将中蜂OBPs与ORs分别与抗螨相关的候选配基进行功能研究和嗅觉结合反应测试。结果显示，发现并克隆到一个中蜂触角Ors表达丰度较意蜂最高的嗅觉受体，即AcerOr9，其表达丰度（RPKM=103.8）要远大于意蜂同源基因（RPKM=14.6），另外对中蜂7个独特的气味结合蛋白基因OBP1,2,10,11,12,14和21成功进行了全长基因克隆和原核表达，利用竞争性荧光结合技术对其进行了功能研究，并对OBP2和OBP11进行了定点突变和功能研究；对OBP11, OBP12进行了免疫血清制备，对中蜂OBP11和AcORco在工蜂触角上进行了免疫共定位研究；成功构建了AcerORco和AcerOR9的真核表达载体构建，分别利用爪蟾卵母细胞-双电极电压钳技术和HEK293-钙成像技术研究了AcerOR9与候选配基的功能模式；最后收集并分析了瓦螨侵染中蜂蛹的挥发物，并完成了中、意蜂工蜂对候选配基的触角电位反应。本研究为继续研究中蜂的嗅觉生理机制奠定基础，并已将部分研究结果整理成13篇论文正式发表（另有3篇SCI论文在投稿中），包括8篇SCI论文（总IF达到23.85）、1篇EI论文和3篇国内一级论文，另外培养硕士生5名。基本完成项目预期所需达到的指标。