嗅觉受体在苜蓿盲蝽趋花行为中的功能研究

Floral odors play critical roles in preference behavior of insects to flowering plants, and olfactory receptors (ORs) play important roles in chemoreception of these odor compounds. Exploration of the OR's function in chemical communication could help us clear insect olfactory identification mechanism, and provide a new strategy for insect pests management by regulating the behavior of odor perception. In the present work, the functional floral odors of cotton which were involved in preference behavior of alfalfa plant bug Adelphocoris lineolatus to flowering plants were identified from by using the GC-EAD and insect olfactometer. Based on the constructed antennal transcriptome of A. lineolatus early, the OR genes of alfalfa plant bug will be identified by bioinformatics methods and RACE technique. Then,the recombinant OR proteins will be expressed in Xenopus oocytes system, an immunocytochemical localization method will be carried out to locate the proteins position in antennal sensilla. By two-electrode voltage-clamp assays, the binding relationships between different recombinant ORs and selected floral odors will be investigated. In addition, a RNAi experiment will be conducted to silence the expression of key OR genes, and by qRT-PCR method to detect silence effects. Using GC-EAD and "H" tube trials detect the silence of candidate OR genes impacts on electrophysiological and behavioral responses of A. lineolatus to cotton floral odors. Thus, we could clear the functional differentiation of ORs in preference behavior of alfalfa plant bug to flowering plants. In generally, clarifying the function and molecular mechanism of ORs in A. lineolatus olfactory perception and exploring the RNAi regulation of insect chemical senses could provide a theoretical basis for design of insect olfactory behavior control agents by using ORs as a target.

寄主植物花香气味物质在昆虫趋花行为中具有重要作用，昆虫嗅觉受体(ORs)在识别该类信息化合物中发挥重要功能。深入研究活性花香气味物质及ORs在识别该类物质中的功能，不仅有助于阐明植食性昆虫趋花行为的嗅觉机制，还可为通过调控害虫嗅觉识别进行害虫治理提供新策略。本申请以苜蓿盲蝽为研究对象，利用顶空法收集棉花花香气味物质，结合GC-EAD和昆虫嗅觉仪鉴定具有电生理及行为活性花香气味物质。在前期触角转录组测序的基础上，通过生物信息学和RACE技术鉴定OR基因，利用原位杂交技术和免疫组化方法研究OR基因的表达定位。通过爪蟾卵母细胞表达系统结合双电极电压钳研究OR蛋白和花香气味分子的对应识别关系。利用RNAi技术沉默OR基因表达，结合GC-EAD和昆虫嗅觉仪，检测缺失表达OR基因对苜蓿盲蝽识别花香气味及趋花行为的影响。阐明苜蓿盲蝽趋花行为的嗅觉机制，为昆虫嗅觉行为调控提供理论依据。

苜蓿盲蝽偏好寄主植物花香气味主要包括：月桂烯、DMNT、罗勒烯、β-榄香烯、大香叶烯-D芳樟醇和癸醛等，还有一些特殊挥发物如蘑菇醇、5-甲基-3-庚酮、马鞭烯酮、2-庚酮等。这些花香气味能引起苜蓿盲蝽电生理和行为趋向反应。鉴定苜蓿盲蝽气味受体OR基因88个，味觉受体GR基因4个，感觉神经元膜蛋白SNMP基因4个，大多数ORs在苜蓿盲蝽触角中高表达。AlinOR59与co-receptor AlinORco在雌、雄虫触角中均有共表达，且定位在毛型感器下方的感觉神经元中。AlinOR13和AlinOR33在在苜蓿盲蝽触角长毛形感器中特异性表达。重组表达了AlinOR2、AlinOR27、AlinOR28、AlinOR13、AlinOR33和AlinOR59。电压钳记录显示，AlinOR2对4种棉花花香挥发物：月桂酸乙酯、十三烷醇、十二醇以及癸醛具有明显反应，其中对癸醛的反应最灵敏。AlinOR13 和AlinOR33对苜蓿盲蝽的性信息素组分有反应。AlinOR27可与十二醛和十二醇有反应，AlinOR28对3种棉花花香挥发物有反应，其中对水杨酸甲酯反应值最大。AlinOR59结合谱广泛，对丙烯酸丁酯、水杨酸甲酯和4-乙基苯甲醛具有明显的电生理反应。另外，AlinOR59对苜蓿盲蝽性信息组分丁酸乙酯具有显著的电生理反应。综上，AlinOR2、AlinOR27、AlinOR28、和AlinOR59可在苜蓿盲蝽搜寻定位花期寄主棉花过程中发挥重要感受功能，参与苜蓿盲蝽趋花和转移为害行为。而AlinOR13、AlinOR33和AlinOR59也涉及苜蓿盲蝽性信息素识别，可在雄虫寻找雌虫过程中发挥重要功能。RNA干扰AlinOR2和AlinOR33的结果表明，靶标基因被抑制85%以上，注射dsRNA的苜蓿盲蝽分别对癸醛和性信息素组分反丁酸反-2-己烯酯的EAG反应下降50%以上，而且趋向行为显著受影响，表明靶标嗅觉受体在调控苜蓿盲蝽趋花行为和性信息素识别过程发挥双重作用。.在Insect Molecular Biology, Journal of Chemical Ecology和 Journal of Insect Physiology等学术发表SCI论文21篇，中文核心期刊论文6篇，授权专利1项。