线粒体呼吸链蛋白参与果蝇细胞凋亡调控的机制研究

细胞凋亡在生物体的免疫应答中起作用，同时也参与重要的发育过程。在植物害虫防控领域，通过探索凋亡途径中的重要调控蛋白，可为研究和开发新型农药提供理论基础。申请人前期工作中，构建了黑腹果蝇hid凋亡激活基因诱导的受四环素控制的胚胎致死系统，在去除四环素培养基上获得约万分之一的存活后代，大部分存活后代存在母系遗传现象。通过线粒体测序，发现一个共同基因突变位点，该基因参与编码呼吸链复合体Ⅰ的亚基蛋白，暂命名为DMN-1。本课题围绕新发现的DMN-1基因突变，通过线粒体编辑、DMN-1过表达等鉴定突变的DMN-1是否逆转凋亡发生；同时在转染DMN-1、hid等外源基因的S2细胞中测试一系列的细胞凋亡指征，包括ATP含量、活性氧含量、跨膜电位的改变、细胞色素C是否释放等，并结合转录组分析探索果蝇细胞凋亡的分子网络，揭示该突变基因在凋亡调控中的作用和调控方式。

Apoptosis plays an important role in the immune response of organisms, and also participates in important developmental processes. In the field of plant pest control, exploring the important regulatory proteins in apoptosis pathway can provide a theoretical basis for the research and development of new pesticides. In the previous work of the applicant, a tetracycline controlled embryonic death system induced by the hid gene of Drosophila melanogaster was constructed. About one in ten thousand surviving offspring were obtained on the tetracycline free medium, and most of the surviving offspring had maternal inheritance. Through mitochondrial sequencing, we found a common gene mutation, which is temporarily named DMN-1, which encodes the subunit protein of respiratory chain complex I. In this project, we will focus on the DMN-1 gene, identify whether DMN-1 could reverse apoptosis by mitochondrial gene editing and DMN-1 overexpression, and will tansfect S2 cells with DMN-1, hid and their combination, test ATP content, reactive oxygen species content, transmembrane potential changes, cytochrome c release, etc., and conduct transcriptome analysis to explore the molecular network of apoptosis, and to reveal the role of this mutated gene in the regulation of Drosophila apoptosis.