【作者】 杨华；

【导师】 杜连祥；

【作者基本信息】 天津科技大学 ， 发酵工程， 2004， 硕士

【摘要】 本论文对酵母细胞在高温和氧化条件下的应激反应进行了研究，通过低剂量刺激酵母细胞产生活性衍生物—LYCD，并筛选了耐高温酵母突变株，开辟了制备LYCD的新途径。 本课题采用呼吸缺陷和正常的酵母菌，分析线粒体功能在酵母氧化应激和热应激反应中的作用。结果发现，呼吸缺陷酵母抵抗致死剂量氧化剂的能力比正常酵母差，说明线粒体对于细胞抗氧化是必需的。而呼吸缺陷酵母的抗热能力却比正常酵母强，说明线粒体对于细胞抗热具有阻碍作用。通过低剂量的预处理，两种酵母都对相应致死剂量产生适应性，说明适应性的产生与线粒体功能无关。 测定了酵母预处理前后细胞内几种重要抗氧化剂（GSH、SOD、CAT）的变化，结果表明：两种预处理都可以使正常酵母细胞中抗氧化剂含量提高。并分析了正常酵母预处理前后细胞内脂质过氧化物—丙二醛（MDA）的含量，发现两种预处理后，再在致死条件下作用，MDA的含量降低，细胞的存活率也升高。这不仅说明，预处理使细胞抗氧化水平提高，减少了MDA的积累，细胞增加了对氧化和热的抗性，更直接说明热致死与氧化关系密切。 将经过H₂O₂和37℃预处理后的酵母进行破壁，分别得到LYCD₁和LYCD₂，发现两者都对细胞抗氧化具有保护活性。分别对其处理条件进行优化，确定了LYCD₁的最佳处理条件为：0.4mmol／L H₂O₂预处理60min，LYCD₂的最佳处理条件为：37℃预处理30min。 通过紫外诱变筛选了耐高温酵母突变株，发现与正常酵母相比，耐高温突变株对热和氧化都具有很强的抗性。而且耐高温酵母提取物也具有保护细胞抵抗氧化的能力。并分析了正常酵母和耐高温酵母细胞内GSH、SOD、CAT的差异，发现耐高温酵母胞内抗氧化水平高于正常酵母菌株。 对正常酵母预处理前后蛋白的变化进行了双向电泳分析，结果表明，有一些蛋白是H₂O₂和37℃都可以诱导合成的，但也有些蛋白是H₂O₂或37℃特异性诱导合成的。此外，对耐高温酵母的蛋白分析后，发现它与正常酵母相比，产生了很多新的蛋白，其中包括热激蛋白，也应包括与细胞抗氧化有关的蛋白。更多还原

【Abstract】 This study was based on stress response of yeast cell under high temperature and oxidative condition. Live Yeast Cell Derivative (LYCD) was produced by yeast cell, which was pretreated by low dose temperature and oxidative condition. The experiment setup the new method producing LYCD by selecting thermotolerant mutant.With comparative the stress response of yeast and respiratory-deficient strains, the effect of mitochondria under oxidative stress and heat stress were analyzed. Our results indicated that mitochondria! function was required for resistance to oxidative stress in yeast, since respiratory-deficient strains had the less survival than the yeast. But respiratory-deficient strains had the more ability for resistance to high temperature, indicating that it was inhibited by mitochondrial function to resist heat stress. Through pretreating by low dose, yeast and respiratory-deficient strains were able to mount an adaptive response to high temperature and H2O2.It showed that mitochondrial was unrequired in adaptive response of yeast.The following conclusions were drawn by testing the change of antioxidants and MDA in yeast cell: pretreating of low dose could increase the content of GSH and the activities of SOD and CAT, could reduce the accumulation of MDA. And these induced the resistance to lethal concentration of 50 and H2O2. Furthermore study showed that heat and oxidative stress may have a common mechanism of damage.. LYCD1 and LYCD2 were produced by breaking up the yeast cell pretreated with H2O2 and 37 C. The results showed that LYCD1 and LYCD2 could protect cell to resist oxidative condition. The optimum conditions of producing LYCD1 and LYCD2 were as follow: the concentration of H2O2 is 0.4 mmol/L, and the treating time is 60min;the temperature is 37 C,and the treating time is 30min.After UV treating, the thermotolerant mutant was selected. And it had the more ability for resistance to high temperature and oxidative condition. The extract of mutant had biological activities, the content of GSH and the activities of SOD and CAT in mutant were higher than the initial strain.Through two-dimensional gel electrophoresis experiment, the proteins were analysed. Some changes were induced by H2O2 and 37 C,which were not made in untreated cells. Other changes were induced by H2O2 or 37 C. hi addition, the proteins of mutant were analysed. The result indicated that lots of new proteins were synthesized, some were heat-shock proteins and others were relative to anti-oxidative.更多还原