葡萄铜胁迫中自噬调控网络及其关键基因的鉴定与功能分析

With the progress of human society and the acceleration of industrialization, people pay more and more attention to soil heavy-metal pollution and the harm it causes to agricultural production safety and human health. Bordeaux mixture has been widely used as a copper fungicides since 1880. Heavy-metal stress has been caused by Bordeaux mixture, along with the sterilization in grapevines. Previous studies in our lab have shown that cell autophagy is involved in copper stress, and time-differential character also found in autophagic activity. However, the details of the regulation network and the function of cell autophagy in copper stress remained unknown. In order to understand the cell autophagy regulation network and the function of key autophagy-related genes, high-throughput sequencing, electron microscope observation, transgenic tobacco system, RT-qPCR, yeast two-hybrid system, and bimolecular fluorescence complementation technologies were employed to study the short-period and long-period regulation networks of autophagy in grapevine leaves under copper stress, observe the plant autophagy phenomenon and physiological change characteristics, and analyze the function of key autophagy-related genes. The above research will help us to complement the mechanism of grapevine leaves in response to copper stress and the theory of plant cell autophagy in response to stress. Meanwhile, it also lays a foundation for improving the ability of plant to resist heavy-metal stress and the application of plants to repair the heavy-metal contaminated soil.

随着人类社会的进步和工业化进程的加快，土壤重金属污染对农业安全生产和人们身体健康造成的危害引起了越来越多的关注。波尔多液是一种长期广泛使用的含铜类杀菌剂，葡萄叶片在喷施波尔多液杀菌的同时，也引起一定的重金属胁迫。本课题组前期研究发现，细胞自噬参与葡萄叶片响应铜胁迫过程，并且具有一定的时间特异性，但其具体的调控网络和功能还不清楚。为了揭示葡萄叶片响应铜胁迫过程中存在的细胞自噬调控网络及其关键基因的功能，拟利用高通量测序、电镜观察、烟草转基因、RT-qPCR、酵母双杂交以及双分子荧光互补等实验手段研究铜胁迫下葡萄叶片细胞自噬的短效和长效调控网络，观察铜胁迫下细胞自噬现象和植株生理变化特征，分析铜胁迫下细胞自噬核心基因的功能。通过以上方面的研究，能够补充葡萄叶片响应铜胁迫机制和植物细胞自噬响应逆境胁迫理论，同时也为提高植物耐重金属胁迫能力和研究植物修复重金属污染土壤的应用奠定一定的基础。

波尔多液是一种长期广泛使用的含铜类杀菌剂，葡萄叶片在喷施波尔多液杀菌的同时，也引起一定的重金属胁迫。基于前期研究发现，细胞自噬参与葡萄叶片响应铜胁迫过程，但其具体的调控网络和功能还不清楚。本项目利用高通量测序、电镜观察、烟草转基因、RT-qPCR、酵母双杂交以及双分子荧光互补等实验手段研究铜胁迫下葡萄叶片短效与长效响应调控网络，分析细胞自噬关键基因在铜胁迫下的功能。研究主要结果包括：系统构建了轻度铜过量胁迫（0.5 mM CuSO4）条件下的葡萄应答调控网络，发现铜离子吸收、转运、螯合与外排，叶绿素合成代谢与光合系统，抗氧化系统三大模块主要参与铜胁迫应答。“碳代谢”、“光合作用-天线蛋白”、“半胱氨酸和蛋氨酸代谢”、“胡萝卜-烯类生物合成”和“氧化还原酶活性”主要在短效响应阶段（0-8h）起作用。“脂肪酸生物合成和代谢”、“谷胱甘肽代谢”、“错配修复”和“植物激素信号转导”主要在长效响应阶段起作用（8-64h）。过表达VvATG8f和VvATG6能够提高拟南芥/烟草对铜胁迫的耐受性。VvATG8f和VvATG6分别与5个和6个蛋白互作，调控多种抗氧化酶/蛋白编码基因的表达。此外，本项目还系统评价了280余份葡萄种质资源耐铜性差异，发现欧美杂交种葡萄耐铜性强于欧亚种葡萄。证实ALA对葡萄铜胁迫具有一定的缓解效应。本项目共发表论文12篇，获批发明专利1件、实用新型专利4件，申请发明专利3件，相关成果获省部级奖励2项。本研究有效补充了葡萄叶片响应铜胁迫机制和植物细胞自噬响应逆境胁迫理论，同时也为提高植物耐重金属胁迫能力和研究植物修复重金属污染土壤的应用奠定一定的基础。