喜树碱诱导甜菜夜蛾细胞凋亡的凋亡因子及通路研究

Apoptosis is caused by the accumulation of genomic alterations regulated by a serials of apoptosis factors through a set of pathway. Transcriptional sequencing (RNA sequencing, RNA-seq) provides insights for a transcription genome-wide understanding apoptosis through analyzing transcriptome structures and sequences. In this project, a combination of RNA-seq and bromouide labeling and sequencing (Bru-seq) is conducted to assess genome-wide changes to apoptosis factors and pathway in Spodoptera exigua Hüber after exposed to camptothecin. We will identify and characterize those apoptosis factors and investigate their function in insect cell apoptosis; assess genome-wide RNA synthesis and stability which suggested a coordinated and complex regulation of apoptosis factor expression at the transcriptional level. Results of these will allow a deeper understanding of apoptosis pathway in S. exigua induced by camptothecin. Furthermore, our studies will provide the new insights to explore environment-friendly pesticides and support the potential use of apoptosis factors in controll of insect pest as special targets.

细胞凋亡以多基因参与的多通路调控机制证明其过程是全基因组参与的复杂反应。转录组测序（RNA-seq测序）技术的应用使对细胞凋亡这一全基因组参与的复杂反应的研究变得简便、容易、快速，被许多研究者用来解决新基因挖掘、转录图谱绘制和信号途径确定等问题。本项目以喜树碱为细胞凋亡诱导物，以甜菜夜蛾中肠细胞系IOZCAS-Spex-II为载体，采用RNA-seq和Bru-seq测序技术，结合Western-Blot，流式细胞仪等技术，研究甜菜夜蛾中肠细胞转录组情况，评价喜树碱对其影响；通过差异表达基因挖掘细胞凋亡因子，明确其功能与作用；检测喜树碱诱导甜菜夜蛾细胞凋亡对RNA合成与稳定的影响，分析凋亡因子在转录水平上的动态变化，绘制喜树碱诱导甜菜夜蛾细胞凋亡通路。本研究结果将为揭示昆虫细胞凋亡机理和喜树碱杀虫活性机制提供重要理论依据，同时对于发现生态安全性特异靶标和环境和谐性农药具重要的应用价值。

细胞凋亡以多基因参与的多通路调控机制证明其过程是全基因组参与的复杂反应。通过本项目实施共筛选到63个凋亡关键基因，分属于Bcl-2 家族、Caspase家族、IAP 家族、TNF家族、AIF家族、p53 家族、Apaf-1和Cyt c等。以果蝇凋亡通路为基础，模拟绘制CPT诱导甜菜夜蛾中肠细胞的凋亡通路图，CPT诱导甜菜夜蛾细胞凋亡过程中存在线粒体内途径和死亡受体外途径，其中线粒体内途径是主要的凋亡途径。凋亡因子Caspase-1和Caspase-5通过裂解产生小亚基的方式活化，与Apaf-1形成凋亡调控中心。凋亡因子SeGrx1通过构建线粒体硫醇信号调节装置，在维持抗氧化系统平衡、保护细胞免受氧化应激和抗CPT诱导的凋亡等方面发挥重要作用。本研究结果为揭示昆虫细胞凋亡机理和喜树碱杀虫活性机制提供重要理论依据，同时对于发现生态安全性特异靶标和环境和谐性农药具重要的应用价值。