自噬在酵母乙醇胁迫中的作用及机制研究

Autophagy plays key roles in aging, death, life-span determination and environmental stresses of yeast, and is usually induced by reactive oxygen species (ROS). Ethanol stress is the most common environmental stress faced by yeast during fermentation, which can lead to damage and death of yeast cells, thus has a negative effect on fermentation. While whether autophagy is involved and what its role is in ethanol stress has not been reported yet. In a preliminary study, the applicant found that the inducing or inhibition of autophagy could increase or reduce the cell death of yeast caused by ethanol stress respectively. ROS production and cell death were accelerated significantly by ethanol stress, and this could be recovered by ROS scavengers, ethanol production were improved obviously at the same time. These results indicated that both autophagy and ROS are closely related to ethanol stress. Based on this, we try to investigate this problem thoroughly by utilizing techniques such as fluorescent microscopy, flow cytometry, Western blot and real-time RT-PCR et al. The following items will be included:(1) Whether autophagy is involved in the ethanol stress and which kind of autophagy is involved; (2)Whether autophagy is induced by ROS and where is the ROS originated from, and which ROS is the key player; (3)How does autophagy affect cell damage and death caused by ethanol stress et al. Results of this study will contribute to understanding the mechanisms of ethanol stress, and provide a theoretical basis for improving ethanol tolerance and ethanol yield of yeast through regulation of autophagy.

自噬在酵母衰老、死亡及逆境胁迫等过程中均具有重要作用，通常由活性氧（ROS）诱导发生。乙醇胁迫是酵母发酵过程中最常见的环境胁迫，可导致酵母细胞损伤及死亡，但乙醇胁迫是否诱导自噬以及自噬在其中的作用，目前尚未见到相关报道。申请者前期研究发现促进或抑制自噬能分别增加或减少乙醇胁迫条件下酵母细胞死亡，且乙醇胁迫显著诱导ROS产生，而清除ROS则可以减少细胞损伤和死亡，提高发酵终点乙醇产率，说明自噬和ROS均与乙醇胁迫密切相关。本项目拟在此基础上，采用荧光显微成像技术，流式细胞技术，Western blot、RT-PCR等手段和方法，以自噬突变体和亲本菌株为材料，系统研究自噬在乙醇胁迫中的作用及相关机制，包括自噬是否发生及自噬类型，是否由ROS诱导，参与诱导的ROS种类及来源，自噬对乙醇胁迫造成的损伤、死亡及功能下降的影响等。本研究可为通过调控自噬提高酵母乙醇耐受性及产率提供理论基础。

在国家自然科学青年基金（N0. 31201409）的资助下，以酿酒酵母BY4742野生型及自噬突变型△ATG1，△ATG8，线粒体自噬突变型△ATG11，△ATG32为对象，研究了乙醇胁迫对细胞巨自噬和线粒体自噬的诱导作用及机制，乙醇胁迫诱导产生ROS的作用及机制，确定了ROS，巨自噬和线粒体自噬在乙醇胁迫条件下在酵母细胞生长、增值和产乙醇能力等方面的作用。并在此基础上，探讨了乙醇胁迫诱导生成的ROS与巨自噬，线粒体自噬之间复杂的调控关系。本研究首次报道了巨自噬及线粒体自噬在酿酒酵母乙醇胁迫中的作用及机制。以上研究为弄清自噬及ROS在乙醇胁迫影响酵母细胞生长，增值及发酵的机制奠定了一定的基础。.（1）明确了乙醇胁迫对细胞巨自噬和线粒体自噬的诱导，证实了其在乙醇胁迫条件下在酵母细胞生长、增值、细胞损伤和产酒精能力中的作用，探讨了乙醇胁迫诱导生成的ROS的来源及作用，并探讨了乙醇胁迫诱导的ROS和细胞巨自噬，线粒体自噬之间复杂的调控关系。.（2）依托该基金资助，目前以通讯作者身份发表核心期刊论文3篇，合作作者身份发表中文核心期刊论文2篇，投稿SCI期刊论文2篇，另有2篇（中英文各1篇）待投稿。.（3）项目负责人到耶鲁大学访问交流。.（4）培养硕士研究生3名。