BmATG5调控变态发育时期家蚕细胞自噬触发凋亡的分子机制研究

Autophagy and apoptosis are the two crucial programmed cell death events during metamorphosis of silkworm. Preliminary data reveals that the bombyx mori autophagy related protein BmATG5 and truncated BmATG5-tN molecules affect autophagy and apoptosis process, respectively, but the elaborate regulation mechanisms of apoptosis are unclear. On this basis, the molecular mechanism of BmATG5-regulated autophagy triggering apoptosis in silkworm will be further studied in this project: （1）to reveal, the key role of BmATG5/BmATG5-tN in regulating autophagy and apoptosis signal pathway in the midgut, fat body, posterior silk gland of bombyx mori and BmE embryonic cell under the stress of exogenous 20E, high concentration Ca2+ , starvation and other factors；（2）to determine the key protein binded to BmATG5 / BmATG5-tN via immunoprecipitation, mass spectrometry and Co-Immunoprecipitation, and further characterize the subcellular localization of BmATG5/BmATG5-tN through immunohistochemistry. Furthermore, the CRISPR/Cas9-based gene editing technique or site direct mutation technology will be used in this project to explore the molecular mechanisms involved by BmATG5/BmATG5-tN and its binded proteins in regulating autophagy and apoptosis during the metamorphosis of silkworm, and thus will provide useful clues for the lepidopteran pest control and regulation of Bombyx mori metamorphosis as well.

自噬和凋亡是家蚕变态发育时期两种重要的细胞死亡方式，前期研究揭示家蚕自噬相关蛋白BmATG5和截短分子BmATG5-tN分别参与自噬和凋亡进程，但调控凋亡发生具体机制不详。在此基础上，本项目将对BmATG5调控家蚕细胞自噬触发凋亡的分子机制展开研究：（1）验证在外源20E、饥饿和Ca2+等因素胁迫下，BmATG5及其截短分子在家蚕中肠、脂肪体、后部丝腺组织和BmE细胞自噬和凋亡信号通路中的关键作用；（2）免疫沉淀BmATG5/BmATG5-tN的结合蛋白，进行质谱分析，和用Co-IP验证其结合，免疫组化分析BmATG5/BmATG5-tN在细胞中的定位；CRISPR/Cas9敲除或是定点突变等技术研究它们的结合蛋白在调控自噬和凋亡发生中的功能，以此揭示BmATG5在调控变态发育时期家蚕细胞自噬触发凋亡的重要作用，为鳞翅目害虫防治和家蚕变态发育调控提供参考依据。

项目通过一序列分子生物学实验技术解析自噬相关蛋白BmATG5及其截短片段BmATG5-tN在调控变态发育时期家蚕细胞自噬触发凋亡过程中的关键作用。结果发现全长的BmATG5能促进家蚕细胞自噬，而截短的BmATG5-tN促进家蚕细胞凋亡，但未奠定到与BmATG5-tN结合的关键蛋白，说明与哺乳动物细胞ATG5/ATG5-tN促细胞自噬和细胞凋亡机制的异同。由于Co-IP没有验证到在细胞凋亡发生时与ATG5-tN的分子，因此促凋亡机制不详，但BmATG5促进细胞自噬和BmATG5-tN促进细胞凋亡过程与哺乳动物相似，为阐明家蚕甚至鳞翅目昆虫细胞自噬和凋亡发生机制及变态发育机理奠定坚实基础。