張雅雯Chang, Ya-Wen臺灣大學:醫學檢驗暨生物技術學研究所李承光Lee, Cheng-KuangCheng-KuangLee2010-05-112018-07-062010-05-112018-07-062008U0001-2808200813493100http://ntur.lib.ntu.edu.tw//handle/246246/182956Saccharomyces cerevisiae一般來說不具有致病性，但近年來陸續感染病例的報告，所以被認為也是一種伺機性感染的病原體。先前的研究指出釀酒酵母菌的臨床菌株多出了能在高溫(42°C)下生長以及假菌絲產生的能力，推測這兩者為可能的致病因子。在台大醫院分離之三個臨床菌株，我們發現當中有兩個具有高溫生長能力以及假菌絲生成能力。本研究目標主要為探討S. cerevisiae的高溫生長能力這個致病因子。先前研究指出高溫生長屬多基因調控，可能參與的基因有：MKT1、RHO2、END3，另外也發現在這些基因上若帶有某些SNP會影響到高溫生長的表現型。我們發現具有高溫生長能力的臨床菌株的定序結果，發現在MKT1 (A1057G)和RHO2 (T271G)處的SNP會造成胺基酸改變，而在這些位置上的等位基因屬異型合子(heterozygous)，但沒有高溫生長能力的菌種則無。於是進行四分孢子分離，得到了同型合子的子代。而分離出來的子代中發現只有一株具有和母代一樣強的高溫生長能力，我們將此子代的高溫生長基因(MKT1)送入實驗室菌株中，觀察高溫生長的表現型是否會因此而有所改變，但經此改造的實驗室菌株，其高溫生長能力並未有發現有顯著的變異，這表示只單純改變一個高溫生長基因，並未能夠影響到其表現型。當酵母菌受到高溫壓力之時，細胞內活性氧化自由基會上升，若此時細胞抗氧化壓力的能力不足，細胞就無法存活下去。我們去比較抗高溫的菌株和不耐熱的菌株比較在受到高溫壓力之時其細胞內活性氧化自由基的累積情況，但結果並未發現抗高溫的菌株其活性氧化自由基的累積會比較低。目前我們尚未找到抗高溫生長的關鍵因子，之後仍需進一步的研究。Saccharomyces. cerevisiae has recognized as “generally regarded as safe” organism, but recent evidence indicated that the clinical isolates were virulent and had been implicated in the induction of a disease. The pathogenic isolates exhibit the ability to grow at 42°C and are capable of pseudohyphal growth. Our lab got three clinical isolates(YYC1、YYC2、YYC3) from NTUH . We found there were two of them having the ability of growth at 42°C and pseudohyphal formation. My target is studying the ability of growth at 42°C. The previous report mapped a S. cerevisiae high temperature growth QTL and identified MKT1, END3, and RHO2 as QTGs. There were some important SNPs (ex.MKT1 A1358G、END3 C733T )contributing to HTG phenotype. In our case, we found that the SNPs in YYC1, MKT1 (A1057G) and RHO2 (T271G), are nonsynonymous changes and heterozygous in these two gene locus. Next, we use tetrad dissection, making the homozygous segregants, try to observe the contribution of these SNPs to HTG. After tetrad dissection analysis, we found that there were few segregants(4/60) having the HTG phenotype. Then, we cloned the MKT1, from one of the segregants which had thermo-tolerance, into a mkt1 deletion strain. However, the clone did not change its phenotype. This indicated that the single genotype alternation did not affect the HTG phenotype. When the yeast cells encounter heat stress, the cellular ROS (reactive oxygen species) will be induced. Excess ROS causes damages by attacking DNA and proteins. Our hypothesis was that the thermo-tolerant strains might have more strong ability to clear up cellular ROS. But the experiment result indicated that the thermo-tolerant strains did not have lower ROS level than thermo-sensitive strains. We still do not find the key factor contribution to the ability of HTG.. It needs advanced researches to figure out the mechanism of high temperature growth.總目次目次………………………………………………………………I目次……………………………………………………………III目次………………………………………………………………IV寫表………………………………………………………………VI文摘要…………………………………………………………………1文摘要…………………………………………………………………2論…………………………………………………………………3料方法、菌落尺寸大小試驗……………………………………………7、生長曲線7、四分孢子分離技術8、細胞凝集試驗……………………………………………………8、聚合酶連鎖反應…………………………………………………8、基因定序………………………………………………………9、分子選殖………………………………………………………11、存活率試驗……………………………………………………14、細胞內活性氧化自由基累積的程度…………………………15果、臨床菌株的高溫生長表現型：菌落尺寸大小、生長曲線、存活率…17、以基因定序手法找出可能影響高溫生長的SNP………………18、同型合子子代的高溫生長能力表現型…………………………19、分子選殖：MKT1對於高溫生長能力的影響…………………19、高溫生長能力和細胞內活性氧化自由基累積程度的關連性…21論…………………………………………………………………23表……………………………………………………………………27圖……………………………………………………………………38考文獻………………………………………………………………50錄、實驗用菌種列表………………………………………………54、培養液…………………………………………………………55、DCFDA結構式………………………………………………55目次一 菌種高溫生長表現型：菌落尺寸大小試驗…………………27二 YYC1子代高溫生長表現型：菌落尺寸大小試驗…………28三 A高溫生長基因的SNP位點分析………………………………30三 B胺基酸序列變化分析…………………………………………33三 C具高溫生長能力子代的SNP位點基因型分析………………35四 四分孢子分離實驗分析………………………………………36五 實驗所用的引子………………………………………………37目次一 菌落尺寸大小試驗…………………………………………38二 存活率試驗……………………………………………………39三 在42°C下的生長曲線(YPAD)………………………………40四 細胞凝聚試驗…………………………………………………41五 抽取分子選殖菌株的質體……………………………………42六 利用限制酵素的裁切來確認選殖菌株………………………43七 利用colonyPCR來確認酵母菌轉型作用…………………44八 基因重組實驗的高溫生長表現型：在42°C下的生長曲線(YM-Ura)……………45九 細胞內活性氧化自由基累積的程度：疊圖分析……………46十 細胞內活性氧化自由基累積的程度：溫度的影響…………48十一 細胞內活性氧化自由基累積的程度：受熱時間比較………49application/pdf3206685 bytesapplication/pdfen-US釀酒酵母菌高溫生長活性氧化自由基Saccharomyces cerevisiaehigh temperature growthROShttp://ntur.lib.ntu.edu.tw/bitstream/246246/182956/1/ntu-97-R95424019-1.pdf