转位蛋白TSPO(18kDa)调控飞蝗型变中嗅觉排斥行为的机理研究

Given tyramine and its receptor regulate olfactory repulsion behavior in solitary locusts and gregarious locusts with exposed to isolation, we identified that translocator protein (18 kDa)(TSPO)in outer membrane of mitochondria is down regulated in the brains of solitary locusts that is injected with double strand RNA of tyramine receptor. Thus, we speculate that TSPO may act as one of the downstream targets in tyramine signaling pathway to modulate olfactory repulsion behavior. In this study, we clone the full length of TSPO and make the antibody of TSPO to analyze its spatial and temporal expressions by qRT-PCR and immunochemistry staining. Along with the behavioral analysis in open field and Y-tube olfactometer, we apply RNA interference and pharmacological intervention to explore whether TSPO regulates olfactory repulsion response in solitary locusts and whether TSPO performs its function as one of the downstream targets of tyramine signaling pathway. Moreover, we choose RNA-seq transcriptome and computational analysis to detect the gene expression profiling influenced by TSPO knockdown in solitary locusts. The metabolomics analysis is also applied to explore the metabolites that differentially change in the brains of solitary locusts with TSPO knockdown. Then qRT-PCR and behavioral assay are applied to validate the results from transcriptome and metabolome analysis. This study firstly explore the function and mechansim of TSPO in regulating olfactory repulsion response, which will provide the solid foundation for the study of locust phase change and behavior-directed pest management.

基于酪胺受体调节散居型飞蝗嗅觉排斥行为的研究基础，将酪胺受体RNA干扰后的散居型飞蝗脑组织进行转录组分析，结果发现线粒体转位蛋白TSPO(18kDa)表达下调。因此，TSPO可能在酪胺受体下游调节飞蝗嗅觉排斥行为。通过克隆TSPO基因全长与制备抗体，采用定量PCR、原位杂交、免疫组化和印迹分析脑中TSPO在飞蝗行为型变中的表达模式。并结合旷场和Y形管行为分析，通过RNA干扰和药理学方法研究TSPO是否调节散居型飞蝗排斥行为，验证TSPO是否作为酪胺受体靶基因调节散居型飞蝗嗅觉排斥行为。采用转录组技术分析TSPO干扰导致的差异基因变化和信号通路，预测表达差异的编码基因和长链非编码RNA互作网络，并进行功能验证。利用代谢组学技术分析TSPO干扰引起的代谢产物变化，寻找调控嗅觉行为的化合物，首次初步阐明TSPO调节嗅觉排斥行为的分子机制，为飞蝗行为型变机理研究和害虫行为导向性防控奠定理论基础。

转位蛋白(translocator protein, TSPO, 18kDa) 最初被认为是苯二氮卓受体，主要和神经心理疾病、神经退行性疾病和神经疾病损伤影像诊断有关。但是对TSPO蛋白调节行为的功能和机制研究不清楚。在本项目中，发现飞蝗TSPO蛋白表现出保守的特点并且在飞蝗体内高表达。TSPO mRNA和蛋白表达水平在散居型飞蝗脑中高于群居型飞蝗，但是TSPO mRNA和蛋白水平在飞蝗群居化和散居化过程中表达稳定，没有差异，说明TSPO可能和散居型飞蝗的行为表型有关。功能分析发现散居型飞蝗脑中TSPO干扰后，其嗅觉排斥行为反应的比例降低。散居型飞蝗脑中酪胺受体干扰后的转录组高通量测序分析发现TSPO mRNA表达水平显著下调。而定量PCR和免疫印迹发现酪胺受体干扰和酪胺注射后分别上调和下调TSPO mRNA和蛋白水平的表达。进一步的功能研究发现散居型飞蝗TSPO RNA干扰抑制了酪胺受体对飞蝗嗅觉排斥行为的调节。而在群居型飞蝗中，TSPO RNA干扰抑制了酪胺诱导群居型飞蝗表现出嗅觉排斥行为。这个项目揭示了酪胺通过TSPO蛋白调节嗅觉排斥行为的新机制，为深入研究飞蝗嗅觉排斥行为机理研究提供理论基础。