绿盲蝽性信息结合蛋白PBPs的功能分化及RNAi研究

Insect pheromone binding proteins (Pheromone binding proteins, PBPs) are a class of olfactory protein expressed in the pheromone-specific sensilla trichodea of antennae, and play important roles in pheromone chemoreception and finding mates behavior. Exploration of the PBP's function in pheromone communication could help us clear insect olfactory identification mechanism, and provide a new strategy for insect pests management by sex pheromone communication behavior regulation. Based on the constructed antennal cDNA library of Apolygus lucorum early, antennal-specific expressed PBP genes of A. lucorum will be identified by bioinformatics methods and real-time quantitative PCR technique. And also, an in situ hybridization technique will be conducted to observe the location of PBP genes in antennal sensilla at mRNA levels. After obtain recombinant PBPs and specific antibodies, an immunocytochemical localization method will be carried out to locate the proteins position in antennal sensilla. By fluorescence binding assays, two-phase binding assays and SPR technologies, the relationships between different recombinant PBPs and sexual pheromones molecules will be investigated. Thus, we could clear the functional differentiation of PBPs in A. lucorum. A RNAi experiment will be conducted to silence PBP genes expression, and by qRT-PCR method to detect silence effects. Using GC-EAD and "Y" tube trials detect the silence of PBP genes impacts on electrophysiological and behavioral responses of A. lucorum to sex pheromones. In generally, clarifying the function and molecular mechanism of PBPs in insect olfactory perception and exploring the RNAi regulation of insect pheromone reception could provide a theoretical basis for selection and design of target insect olfactory behavior control agents.

昆虫性信息素结合蛋白(PBPs)在昆虫感受性信息素及寻找配偶中发挥重要作用。深入解析PBPs在性信息素通讯中的功能不仅有助于阐明昆虫嗅觉识别机制，还可为通过调控害虫信息素通讯行为进行害虫治理提供新策略。本申请以绿盲蝽为研究对象，在前期构建触角cDNA文库的基础上，通过生物信息学和荧光定量PCR方法鉴定PBP基因，利用原位杂交技术在mRNA水平研究PBP基因的表达定位。表达重组PBP蛋白，采用免疫组化手段在蛋白水平对PBPs进行免疫定位。通过荧光结合实验、双相结合实验和表面等离子共振分析研究PBP蛋白和性信息素分子的对应识别关系。利用RNAi技术沉默PBPs基因表达，通过qRT-PCR检测沉默效果，结合GC-EAD和昆虫嗅觉仪检测缺失表达PBPs基因对绿盲蝽感受性信息素的电生理及交配行为的影响。阐明绿盲蝽PBPs的功能分化，为RNAi靶向调控昆虫信息素感知及设计昆虫嗅觉行为调控剂提供依据。

采用转录组测序技术对绿盲蝽触角高通量测序，共计获得38个完整OBP序列全长,基本覆盖了绿盲蝽全部气味结合蛋白基因。在38个鉴定的OBPs中，17个OBPs在雌雄触角表达水平显著高于其他组织，其中有7个在雄性触角表达水平显著高于雌性触角，有7个在雌性触角表达水平显著高于雄性触角；有3个OBPs在口器表达水平显著高于其他组织；有7个OBPs在头部表达水平显著高于其他组织；有5个OBPs在足部表达水平显著高于其他组织；有1个OBPs在腹部表达水平显著高于其他组织；有1个OBPs在翅部表达水平显著高于其他组织。筛选AlucOBP7，AlucOBP8，AlucOBP11，AlucOBP13，AlucOBP17，AlucOBP29，AlucOBP36作为潜在的PBPs。竞争结合试验结果表明：AlucOBP7能够结合盲蝽类性信息素类似物4-氧代-反-2-己烯醛及植物挥发物2-己酮和水杨酸甲酯，AlucOBP8只结合植物挥发物葎草烯和十二醛，AlucOBP5、AlucOBP13结合花香气味β-紫罗酮，AlucOBP14、AlucOBP17、AlucOBP22能够结合盲蝽类性信息素类似物丁酸辛酯、己酸己酯和棉花挥发物十二醛、β-紫罗酮，AlucOBP36、AlucOBP37、AlucOBP38结合棉花次生代谢物质单宁及棉酚等。鉴定AlucOBP7、AlucOBP14、AlucOBP17、AlucOBP22为绿盲蝽PBPs。RNA干扰AlucOBPs和非典型气味受体ORco结果表明，注射siRNA 的绿盲蝽对性信息素组分反-2-己烯醛和丁酸反-2-己烯酯的EAG反应显著下降。室内大量行为学观察发现，绿盲蝽只有在寄主植物挥发物的大背景下及取食寄主植物的前提条件下，人工合成性信息素对绿盲蝽雄虫才具有吸引效果。这一发现与本研究绿盲蝽性信息素结合蛋白同时行使结合植物挥发物功能提供了行为学证据。室内测定了绿盲蝽对66种气味化合物触角电位（EAG）反应，发现与候选PBPs结合较强的2种棉花挥发物和4种盲蝽科性信息素都能引起很强的EAG反应。这些功能化合物可以作为绿盲蝽嗅觉调控剂备选组分应用到田间绿盲蝽趋性行为控制策略中。. 项目在BMC Genomics, Journal of Chemical Ecology等期刊发表SCI源论文23篇，中文核心期刊论文6篇，授权发明专利2项。