保幼激素-钙信号通路调控飞蝗卵泡上皮细胞Na+/K+-ATPase的分子机制

The uptake of vitellogenin by the oocyte via intercellular spaces (patency) between the ovarian follicular epithelial cells is an necessary stage during the insect oogenesis. In various insects, Juvenile hormone (JH)-calcium signaling pathway regulates ovarian patency which is dependent on the action of Na+/K+-ATPase, whereas the related mechanism is unclear. In regard of that, we will focus on the analysis of the molecular mechanism involved in the JH-triggered Na+/K+-ATPase activity through calcium signaling pathways in Locusta moratoria. RNA interference (RNAi) will be used to screened calcium signal transduction factors (i.e. Protein Kinase C, PKC and Ca2+/calmodulin-dependent protein kinase II, CaMKII) involved in the activation of Na+/K+-ATPase through calcium signal pathways during JH-induced ovarian patency. Posttranslational modification will be analyzed by mass spectrometric analysis for the activation mechanism of PKC, CaMKII and Na+/K+-ATPase involved in the JH-triggered ovarian patency in L. moratoria. Subsequently, the molecular mechanism of Na+/K+-ATPase activated by PKC and CaMKII will be identified by site-directed mutagenesis, RNAi and Co-Immunoprecipitation (Co-IP). Finally, the molecular mechanism of Na+/K+-ATPase activated by JH-calcium signaling pathways will be clarified. The key signaling pathways researched in this study will supply the theoretic support for pest control by reproductive mechanism.

通过卵巢滤泡上皮细胞的间隙（patency）吸收卵黄蛋白原是昆虫卵黄发生的关键环节。大多数昆虫中，保幼激素（JH）-钙信号通路在Na+/K+-ATPase依赖的卵巢patency调控中起到关键的作用，然而分子机制不明确。基于此，本项目拟以飞蝗JH - patency开展工作，旨在分析JH钙信号通路激活Na+/K+-ATPase的分子机制。利用RNA干扰（RNAi）筛选出通路中激活Na+/K+-ATPase的钙信号转导因子--- PKC，CaMKII。通过质谱数据鉴定通路中PKC、CaMKII以及Na+/K+-ATPase翻译后修饰位点，分析这些信号转导分子的活化机制。而后运用定点突变、蛋白亚细胞定位、RNAi、免疫共沉淀等技术，最终阐明PKC、CaMKII激活Na+/K+-ATPase的分子机制。本项目研究中的关键信号通路的阐明，为利用生殖分子机制作为害虫绿色防控提供理论支撑。

保幼激素JH调控多数昆虫卵黄发生，包括上调昆虫卵黄发生期脂肪体卵黄原蛋白Vg表达以及促进卵巢对Vg吸收。研究表明，卵母细胞外周的滤泡上皮细胞间隙（patency）在控制卵巢吸收Vg过程中起到了关键的调控作用，并且Na+/K+-ATPase在控制patency形成过程中起到至关重要的作用，但是其中JH的调控机制不明。本项目以飞蝗（Locusta migratoria）作用研究对象，通过对调控patency形成调控中的Na+/K+-ATPase活化分子机制进行解析，并同时对该调控过程中的JH信号转导通路进行探究，包括膜上受体、第二信使系统以及蛋白激酶。研究结果表明，JH能够通过触发G蛋白偶联受体（GPCR）、受体酪氨酸激酶（RTK）、磷脂酶C（PLC）、IP3R以及Ca2+，激活蛋白激酶C（PKC）信号通路，继而诱导Na+/K+-ATPase α亚基Ser8位点磷酸化修饰，从而激活Na+/K+-ATPase促进patency形成，最终调控卵巢吸收Vg。该研究成果丰富了Na+/K+-ATPase活性调控机制以及在昆虫生殖发育中的功能研究，并且信号通路的阐明推进了昆虫JH研究、特别是JH快速信号转导通路调控昆虫卵黄发生相关机制研究进展；同时能够为害虫绿色防治提供研究基础。