性信息素结合蛋白在识别TypeII型茶尺蠖性信息素中的功能研究

Species-specific sex pheromone recognition is crucial to mate finding and a successful reproduce in moth species, and its molecular mechanism is always the research hot topic in chemical ecology, in which pheromone binding proteins (PBPs) as the first target protein to interact with sex pheromones in the olfactory sensillum, which play key roles in binding and discriminating different pheromone components. To date, the physiological functions of PBPs related to Type II sex pheromones remain unclear in comparison to various information from PBPs in the recognition of Type I pheromones. In the current project proposal, we mainly focus on the physiological functions of PBPs from Ectropis obliqua Prout, one of the most serious tea pests secreting Type II sex pheromones. First, based on the previously successful identification of EoblPBPs gene sequences, the expression profiles among different tissues in male and female moths and during different developmental stages will be analyzed. Secondly, the chemosensillum localization of PBPs in pheromone-sensitive sensilla will be investigated applied to use the technical of single sensillum recording, in situ hybridizations and immunocytochemistry. Finally, using ligand-binding assay, site-directed mutagenesis in vitro in combination with RNA interference in vivo to evaluate the physiological functions of PBPs in perception of sex pheromones in E.obliqua. Our study answers the call by addressing further research on olfaction molecular mechanism in the moth species secreting Type II sex pheromones, which should be lay the foundation to explore RNAi with PBPs as target proteins for mating behavior regulation, eventually, contributes towards development of new technical solutions in pest management.

性信息素识别是蛾类昆虫求偶定位、交配繁衍过程中的重要环节, 其分子机制一直是昆虫化学生态学领域的前沿热点。性信息素结合蛋白PBPs是昆虫嗅觉感器中第一个与性信息素分子相互作用的靶标蛋白，对性信息素分子的识别至关重要。目前，感知TypeI型性信息素的PBPs研究较为系统，但感知TypeII型性信息素的PBPs研究相对匮乏。本申请拟以重要害虫茶尺蠖为对象，基于已成功鉴定PBPs基因的研究基础，拟先分析PBPs的表达谱；进一步应用原位杂交、免疫组化和单感器记录技术研究PBPs在雄蛾性信息素敏感感器中的表达分布。通过体外配体结合、定点突变和活体水平RNAi技术系统的阐明PBPs在识别茶尺蠖TypeII型性信息素中的生理功能。研究结果有助于揭示蛾类昆虫感受TypeII型性信息素的分子机制，也可为利用PBPs靶向设计合成高效的嗅觉干扰剂调控茶尺蠖求偶行为，进而发展新型害虫防控技术提供科学依据。

蛾类昆虫性信息素按其结构可主要分为两类：C10-C18不饱和脂肪醇、醛和醋酸酯 (Type I)；C17-C23多元不饱烃及其环氧衍生物 (Type II)。性信息素结合蛋白pheromone-binding proteins (PBPs) 对于Type I性信息素的识别至关重要，而其对Type II 性信息素的识别未见报道。茶尺蠖是茶树最重要的鳞翅目害虫，其主要雇用3种Type II性信息素组分进行求偶交配。本项目综合运用基因鉴定、荧光定量、原位杂交、竞争性结合实验等技术详细研究了PBPs在识别茶尺蠖Type II性信息素中的生理功能。主要取得如下结果：1)分析茶尺蠖幼虫及成虫雌雄蛾触角转录组，鉴定4个茶尺蠖性信息素结合蛋白EoblPBP1-4。2)荧光定量qPCR结果显示EoblPBP1-4均高表达于雄蛾触角；EoblPBP1-4表达模式与茶尺蠖雌雄交配的节律高度相关，EoblPBP1-4表达高峰均出现在雄蛾羽化当天至羽化后第2天，且交配行为并不显著影响EoblPBP1-4的表达。3)扫描电镜结果显示茶尺蠖雌雄蛾触角感器形态及分布明显不同，毛形感器 Str I主要分布在雄蛾触角，毛形感器Str II则主要分布在雌蛾。4)透射电镜结果表明雄蛾触角特异的Str I是典型的嗅觉感器，依据感器神经元树突数目可以分为不同亚型，推测其是茶尺蠖性信息素敏感的感器。5)荧光原位杂交结果表明，EoblPBP1-2，EoblPBP4均特异表达在雄蛾触角毛形感器Str I，EoblPBP3则同时表达于Str I 和Sba。6) 成功表达EoblPBP1-2，EoblPBP4，体外功能实验证实EoblPBP1-2和EoblPBP4均能识别结合茶尺蠖性信息组分。相比于Z3, Z6, Z9-18: H，EoblPBP1-2偏好识别环氧类性信息素组分Z3, epo6, Z9-18: H及Z3, epo6, Z9-19: H。EoblPBP4可同时识别茶树绿叶挥发物茶尺蠖性信息素组分。本项目重点阐明了EoblPBPs的组织表达谱及感器亚细胞定位，明确了EoblPBPs与茶尺蠖性信息素的对应识别关系，为进一步研究PBP在识别蛾类Type II性信息素中的功能，设计以PBP为靶向的Type II性信息素类似物进行茶尺蠖的田间绿色防治提供理论依据。