活性氧在酿酒酵母乙醇胁迫中的作用
助人为乐酿酒酵母是一种传统的乙醇生产微生物，通常用于乙醇燃料生产和酿造业，但过量的乙醇会抑制细胞的生长和活性，对酵母细胞产生毒害作用。ROS〔Reactive Oxygen Species〕是一类性质爽朗的小分子化合物，在多种逆境胁迫中期起重要作用，然而否在乙醇胁迫中起重要作用尚无定论。
为了解ROS在乙醇胁迫中的作用及机制，本文以酿酒酵母为材料，检测了乙醇胁迫对酵母细胞超氧阴离子〔O2·-〕含量、过氧化氢〔H2O2〕含量、酵母细胞生长状况、膜完整性、线粒体膜电位以及脂质和蛋白质损害的阻碍。
实验结果说明，乙醇能够抑制对数生长期酵母的生长，导致膜完整性受损、线粒体膜电位降低、O2·- 和H2O2水平增加、脂质过氧化物MDA含量增加。加入外源还原型谷胱甘肽〔GSH〕，蛋氨酸〔Met〕，N-acetylcysteine〔N-乙酰半胱氨酸，NAC〕picture怎么读后，能够提高酿酒酵母细胞存活率及膜完整性，线粒体膜电位、降低O2·- 和H2O2水平。添加GSH，Met，NAC到发酵培养基中，检测发酵后乙醇含量发觉，正常组为7.3%，Met，NAC能够将乙醇产
率分别提高到8.62%和8.56%，而GSH只有6.35%。以上结果说明乙醇能够诱导O2·- 和H2O2spider是什么意思等ROS的产生，导致氧化胁迫，从而造成酵母细胞发生膜质过氧化，生长受到抑制。而GSH，Met，NAC能够清除过量的ROS，降低乙醇胁迫引起的酵母损害，其中Met和NAC尽管能够提高乙醇产率，然而只有NAC处理在统计学上具有差异性。
为进一步研究ROS变化的机制，采纳自噬抑制剂3-MA和自噬诱导剂雷帕霉素(rapamycin)处理乙醇胁迫条件下的酵母细胞，检测对O2·- 和H2O2水平，菌落形状以及膜完整性的阻碍。结果说明，3MA能够有效降低O2·-英语能力竞赛 和H2O2水平，降低乙醇对酵母菌落的抑制，提高膜完整性，而雷帕霉素进一步加剧对菌落的抑制，降低膜完整性，提高O2·- 和Hstuck in my heart2O2水平。说明自噬是阻碍ROS水平的机制之一，它通过调剂ROS参与酿酒酵母乙醇胁迫损害。
关键词：酿酒酵母；乙醇胁迫；自噬；ROS
Abstract
Saccharomyces cerevisiae is a strain ud to produce ethanol and is applied in the production of ethanol fuel and brewing industry.Saccharomyces cerevisiae is a tranditional yield ethanol strain, but is nsitive to the high concent of ethanol. Excessive accumulation of enthanol could inhibit the growth and activity of cells, induce reactive oxygen species，thereby lead to a damage to yeast cells. Autophagy is an important degradation pathway for maintaining the balance and matter cycle in eukaryotic cells. Although we have a certain amiynt rearch on ethanol stress, the mechanism of ethanol stress in Saccharomyces cerevisiae, which ROS plays a leading role under ethanol stress, whether autophagy is involved in ethanol strees and what role autophagy has in ethanol. The above problems are not yet clear.
To understand which ROS has an important role in ethanol stress, Saccharomyces cerevisiae from Angel Yeast was as the experimental material in this work, we studied the effect of reactive oxygen species (ROS) on Saccharomyces cerevisiae growth and fermentation under ethanol stress, through detecting physiological indicatiors including cell growth, the membrane integrity, superoxide anion(O2·-2013江西高考英语) conctent, hydrogen peroxide
(H2O2)content, malondialdehyde(MDA), carbonyl content and so on. The result showed that ethanol could inhibit the growth of yeast cells in the logarithmic pha, damage the membrance integrity, ri the levels of O2·- and H2O2 , increa the content of MDA (the product of Lipid peroxide). We ud exogenous Glutathione (GSH), Methionine (Met) and N-acetylcysteine (NAC) treated ethanol stresd Sacchayomycescerevisiae respectively, and found that they could reduced the level O2·-qna and H2O2, incread cells activity and decread the concent of MDA. This indicates tha ethanol couldinduce the generation of O2·- and Hjason earles2O2, leding to the oxidative stress, resulting in the damage to yeast cells. When the growth inhibited rious, ethanol even could lead to the death. GSH and Met, NAC could clear the excess of ROS, reduce the yeast damage caud by ethanol stress and improve the ethanol yield.
女友落难记
In order to understand whether autophagy was involved in the ethanol stress in the S. cerevisiae as well as it was related with ROS, we ud 3-MA (autophagy inhibitor) and rapamycin (autophagy inducer) to treat ethanol-stresd Saccharomyces cerevisiae, the
membrane integrity was by PI staining, the level of O2·- and H2O2 was detected by ROS fluorescent probe DHE and DCFH-DA staining. The results showed that 3-MA could reduce the inhibition of ethanol on yeast cells, improve the membane integrity, reduce the levels of O2·- and H2O2. But rapamycin could further deepen the colony inhibition, reduce the membrane integrity, improve the levels of O2·- and H2O2. This indicates that autophagy involves in Saccharomyces cerevisiae ethanol stress by regulating the level of ROS, and plays a role of maintaining cell survival in ethanol-stresd Saccharomyces cerevisiae.
