斜纹夜蛾细胞凋亡体的鉴定和功能研究

The formation of apoptosomes is an important event in mitochondrial apoptosis signaling pathway, apoptosomes have an important role in apoptotic signal transduction and mediated caspase cascade. However, in Lepidoptera, apoptosome formation, composition and function of the apoptosome have not yet been determined or the lack of experimental evidence. Based on previous studies which has proved that insect cell apoptosis involved in the process of release of cytochrome c and the caspase-3 ,caspase-9 activation were depended on cytochrome c in the apoptosis induced with baculovirus and Apaf-1 gene had been cloned in Spodoptera litura Sl-1 cells,in this project, We will identify the formation of apoptosome and apoptosome composition and properties; determine cytochrome c and Apaf-1 binding and relationships by bimolecular fluoresence complementation,determine the locazation and assembly dynamics of Apaf-1 labeled with mGeos-2 or Skylan-S by CLSM and PALM ，examine the effects of RNAi of Apaf-1 and cytochrome c expression on apoptosis and the impact of caspase activity and caspase cascade respectively , test the assembly of Apaf-1 and relation among cytochrome c and caspases in vitro and analyze the function of the apoptosomes’ core component , Apaf-1 .The results will reveale the fucntion of apoptosome in apoptosis in Spodoptera litura cells. The proposed study will have important significance for describing systematically the role of apoptosome in insect apoptotic signal transduction and the mitochondrial signaling pathway network in Lepidoptera.

凋亡体的形成是细胞凋亡线粒体信号通路的重要事件，凋亡体在凋亡信号转导中具有重要的作用。但鳞翅目昆虫细胞凋亡过程中凋亡体形成、凋亡体的成分和功能尚未确定。本项目拟在已经证明鳞翅目昆虫斜纹夜蛾细胞凋亡过程中释放细胞色素c、克隆Apaf-1基因等基础上，分析鉴定斜纹夜蛾Sl-1细胞凋亡体的形成、凋亡体组成；BiFC等技术测定细胞色素c与Apaf-1的结合与相互作用；用改造的mGeos-2、Skylan-S标记Apaf-1，共聚焦、PALM等成像技术分析其定位与聚合动态；RNAi分别干涉细胞色素c和Apaf-1表达对细胞凋亡、caspase-9活性以及caspase级联的影响; 离体测定Apaf-1与细胞色素c、caspase关系;解析凋亡体核心组分Apaf-1基因的功能等，揭示斜纹夜蛾细胞凋亡过程中凋亡体的功能。本项研究对于阐述凋亡体在鳞翅目昆虫细胞凋亡信号转导中的作用具有重要意义。

项目摘要： 凋亡体在线粒体凋亡途径中具有重要的作用，双翅目昆虫果蝇研究显示，在线粒体凋亡信号途径中形成无细胞色素C的凋亡体，但是鳞翅目昆虫有关凋亡体的构成与功能尚不清楚。本项目较为深入研究了鳞翅目昆虫斜纹夜蛾凋亡体的构成，各组分之间的关系以及功能，项目研究取得重要研究结果和成果。1）凋亡体抑制剂FSBA能够抑制细胞斜纹夜蛾细胞凋亡；2）分析鉴定了鳞翅目昆虫斜纹夜蛾细胞中存在构成凋亡体apaf-1、Sl-caspase-5（caspase9同源物）和细胞色素C三种组分；3）Slapaf-1通过调节起始caspase Sl-caspase-5活性介导caspase级联反应；4）Slapaf-1以散在形式定位与细胞质中，但当细胞发生凋亡时，它与细胞色素C结合聚集成复合体结构；5）RNAi 沉默斜纹夜蛾Apaf-1 基因的表达，显著抑制或降低细胞凋亡水平，这说明Apaf-1在细胞凋亡过程中具有重要的作用；6）斜纹夜蛾Apaf-1过表达能够促进昆虫细胞凋亡，但该基因过表达并不直接诱导细胞凋亡，这是由其在线粒体信号通路中的作用方式决定的；7) 斜纹夜蛾细胞色素c和Apaf-1在细胞中共定位，双分子荧光互补实验进一步证明在鳞翅目昆虫细凋亡过程中，存在细胞色素C与Apaf-1之间产生相互作用，表明细胞色素c 是Apaf-1 的辅助因子。本项目提的供斜纹夜蛾凋亡体三种组分的鉴定；凋亡体核心组分Apaf-1基因的功能解析；各组分之间的关系和作用分析等信息资料，揭示在鳞翅目昆虫细胞凋亡过程中形成类似于哺乳动物细胞的凋亡体，也即从线粒体中释放的细胞色素C与Apaf-1结合形成复合体，形成的凋亡体传递凋亡下游事件。这些结果对于进一步探索与阐释鳞翅目昆虫细胞凋亡信号传递具有十分重要的意义。