臺灣博碩士論文加值系統

本實驗為了研究YDL100c基因產物對S. cerevisiae生長於氧化壓力下的影響，將野生株（WT）及YDL100c基因缺失的突變株（KO）細胞，培養於30oC並添加氧化劑tert-butyl hydroperoxide（t-BOOH），6小時後，針對細胞內的抗氧化防禦系統的表現、分子氧化程度、膜脂質過氧化程度、trehalose的累積及glutathione（GSH）的含量進行分析。初步結果為添加t-BOOH後，KO菌株的生長較WT菌株落後且落後的原因為細胞死亡，而無論WT或KO菌株，細胞內分子氧化程度皆比未添加氧化劑時減少，但膜脂質過氧化的程度皆稍微增加，顯示t-BOOH雖未對細胞內分子造成氧化性的損傷但對細胞膜仍有影響。t-BOOH對WT與KO菌株的catalase活性皆有促進作用，但造成KO菌株細胞內的GSH含量較WT菌株少而trehalose的含量則較WT菌株多。當利用RT-PCR分析相關的防禦基因的mRNA表現量時，對KO菌株而言，添加t-BOOH後，GSH1、CTT1、TPS1、TSL1與NTH1的mRNA表現量均低於WT菌株，而SOD1、TRR1及TRX1的表現則無差異，顯示YDL100c基因的缺失對S. cerevisiae生長於t-BOOH下除了影響general stress response的調控外，亦與specific stress response有關聯。其中，GSH1的表現與細胞內GSH的含量相符，而trehalose代謝相關基因的變化也與細胞內trehalose的含量一致，但是CTT1的表現量卻與catalase的活性分析不符。綜合以上結果推測本實驗中KO菌株細胞死亡的原因與細胞內GSH含量的下降及trehalose的累積最為相關。

To study the role of YDL100c during the growth of Saccharomyces cerevisiae in the presence of oxidant, the wild type strain (WT) and YDL100c disrupted strain (KO) were grown at 30oC for 6 hr after adding 0.25 mM of tert-butyl hydroperoxide (t-BOOH). The cells of both strains were assayed for the expression of anti-oxidant system, trehalose accumulation, intracellular molecular oxidation level, membrane lipid peroxidation, and glutathione (GSH) content. The results show that growth of KO is slower than that of WT and the cause of growth delay is the cell death. The data also show that the molecular oxidation level is lower but the lipid peroxidation of membrane is higher in KO compared with WT in the presence of t-BOOH, indicating that ROS do cause the damage on membrane. Further, analysis of the expression of cellular defense-related genes show that expressions of GSH1, CTT1, TPS1, TSL1, and NTH1 in KO are lower than in WT, but expressions of SOD1, TRR1 and TRX1 have no difference, demonstrating that the deletion of YDL100c in S. cerevisiae affects the general and specific stress response when grown in the presence of t-BOOH. In general, the decrease in CTT1 expression is not consistent with the catalase activity assay, however, decreased expressions of GSH1 and genes involved in trehalose metabolism are consistent with the decreased GSH content and increased trehalose accumulation in KO compared with WT. Therefore, the cause of KO cell death in the presence of t-BOOH is most likely related to the decrease in cellular GSH level and trehalose accumulation.

背景介紹 -------------------------------------------------------------- 1
材料與方法-------------------------------------------------------------- 12
結果 -------------------------------------------------------------- 20
討論 -------------------------------------------------------------- 24
參考文獻 -------------------------------------------------------------- 30
表 -------------------------------------------------------------- 35
圖 -------------------------------------------------------------- 36
附表 -------------------------------------------------------------- 44
附圖 -------------------------------------------------------------- 46

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