草地贪夜蛾Sf-IBM1调控线粒体通路诱导Sf9细胞凋亡机制

细胞凋亡是一种受基因调控的程序性死亡方式，参与昆虫生长发育调控和响应外界刺激等生理过程。线粒体通路被证实参与调控草地贪夜蛾Sf9细胞凋亡，但该通路的调控机制却知之甚少。申请人前期从草地贪夜蛾中克隆了果蝇Reaper同源基因Sf-IBM1，功能分析发现Sf-IBM1定位于线粒体且参与调控线粒体通路诱导Sf9细胞凋亡。在已有基础上，本项目拟采用过表达技术结合线粒体指标分析探究Sf-IBM1调控线粒体的方式；GST pull-down结合质谱筛选线粒体中的Sf-IBM1候选互作蛋白，Co-IP及酵母双杂交验证两者的互作关系及互作方式；互作蛋白dsRNA和Sf-IBM1过表达质粒共转染分析线粒体通路和凋亡指标变化，探究两者互作在线粒体通路调控和细胞凋亡中的功能。预期成果将阐明Sf-IBM1调控线粒体通路诱导Sf9细胞凋亡的机制，进一步丰富鳞翅目昆虫细胞凋亡理论，同时为草地贪夜蛾防控新策略提供基础。

Apoptosis is a conserved and highly regulated process of cell death and serves as a homeostatic mechanism to regulate the development and defense responses of insects to a wide variety of biotic and abiotic stresses. The mitochondrial pathway has been confirmed to be involved in the apoptosis process of Spodoptera frugiperda Sf9 cells, but the regulatory mechanism of this pathway is poorly understood. We previously cloned Drosophila Reaper homologous gene in S. frugiperda and named as Sf-IBM1. Our preliminary results revealed that this protein is localized in mitochondria and involved in mitochondrial pathway regulation and apoptosis induction in Sf9 cells. Based on these previous results, we intend to analyze the mode of Sf-IBM1 that regulates mitochondria first. Then we plan to identify the candidate proteins that interact with Sf-IBM1 in mitochondria through GST pull-down and LC-MS/MS analyses. Co-IP and yeast two-hybrid system would be used for verifying the interaction and interaction modes. Besides, the functions of interaction between Sf-IBM1 and its interacting protein will also be studied by detecting the index of mitochondrial pathway and apoptosis in the cells co-transfected with the plasmid harboring Sf-IBM1 overexpression and the dsRNA of interacting protein gene. The expected results will not only reveal the molecular mechanism of Sf-IBM1 regulating the mitochondrial pathway and apoptosis in Sf9 cells, but also enrich our knowledge on the apoptosis in lepidopteran insects and may lead to new strategies for S. frugiperda control.