鄭秋萍臺灣大學:植物科學研究所陳巧燕Chen, Chiao-YenChiao-YenChen2010-05-112018-07-062010-05-112018-07-062009U0001-0402200911441400http://ntur.lib.ntu.edu.tw//handle/246246/181946生物在正常生長代謝過程中，會產生活性氧化物質(reactive oxygen species, ROS)，做為訊息傳導分子，幫助生物抵抗逆境，順利進行生長發育。適量的ROS可以保護生物，但過量的ROS反而會破壞蛋白質、脂質及DNA，造成細胞損壞，導致細胞死亡。本論文針對酵母菌S. cerevisia兩個基因，利用遺傳、分子及DNA微陣列等研究策略，分析它們在氧化逆境的功能進行研究。首先，YGR021w產物係隸屬具跨物種保守性但功能未知的新穎DUF28蛋白家族；本論文的研究結果發現，此基因在不同氧化逆境下其表現會被抑制，而其剔除株對於氧化逆境的抗性增加，且提早或大量誘導具代表性之已知抗氧化逆境相關基因的表現，而過量表現株的特質則反之；故首次發現DUF28蛋白在酵母菌抗氧化逆境機制中具負面調控。另外，微陣列結果進一步顯示，在YGR021w剔除株中抗逆境相關基因較提早且大量表現，醣類及能量相關基因也大量表現，可能在醣類及能量上能更有效之利用，且其細胞內之氧化還原之平衡，也較WT更具穩定性，顯示YGR021w剔除菌株之耐受性，可能是多種有利因素而得到之結果。其次，本論文也以色胺酸合成酶YGL026c (TRP5)為研究對象，結果發現，此基因在不同氧化逆境下其表現會被誘導，TRP5剔除株對過氧化氫及膜相關逆境之敏感度增加，且在氧化逆境下TRP5剔除株中與抗氧化逆境相關之基因的誘導表現比在WT中較為延遲及減弱。另外，微陣列結果顯示，在正常條件下，TRP5缺失即會造成酵母菌本身與膜構造及抗逆境相關基因之表現下降；而在氧化逆境下，其抗氧化、抗其他逆境及蛋白質降解相關基因之表現也是下降，可能在TRP5剔除株中大量之ROS造成之損壞物質無法藉由蛋白質降解過程移除，且缺乏了移除危機之能力，這些皆可能為造成TRP5剔除菌株對氧化逆境敏感的原因。這些結果顯示，酵母菌在遭受逆境時，需有具正負調控能力的不同的蛋白共同參與並協調，以達到最佳的最後逆境反應結果，以維持其生存。Cells growing aerobically are exposed to reactive oxygen species (ROS) generated during metabolism. These ROS can seriously damage the cell by reacting with cellular components, causing oxidative stress and cell death. The aim of this study is to investigate roles of two S. cerevisiae genes, YGR021w and YGL026c (tryptophan synthase, TRP5), in oxidative stress response by taking genetic, molecular and microarray approaches. Firstly, YGR021w is predicted to encode a protein belonging to a conserved novel protein family, namely DUF28. The results showed: (1) YGR021w expression was repressed by oxidative stress factors; (2) Deletion of YGR021w led to enhanced tolerance to hydrogen peroxide and heat shock stress, and YGR021w over-expression had opposite effects; (3) Stress-induced expression of oxidative response-related genes in YGR021w-deleted strain was faster and stronger compared to the wild-type strain (WT) ; (4) Microarray-based transcriptome analysis revealed enhanced induction of genes involved in oxidative response, carbohydrate metabolism in YGR021w-deleted strain compared to the WT. As the first report, these results together indicated that YGR021w plays a negative role in yeast response to oxidative stress. Secondly, role of TRP5 in oxidative stress has not been elucidated until this study. The results showed: (1) TRP5 expression was induced by oxidative stress factors; (2) Deletion of the TRP5 led to remarkably increased sensitivity to various oxidative stresses and hydrophobic toxic compounds, while complementation rescued some of these defects; (3) Stress-induced expression of oxidative response-related genes in TRP5-deleted strain was much reduced compared to the WT; (4) Exogenous supplement of plant hormone IAA, enhanced tolerance to oxidative stress in the WT and the TRP5-deleted strain; (5) Microarray analysis revealed decreased expression of genes involved in stress tolerance and protein degradation in the deleted strain compared to the WT. Therefore, in addition to possible loss of cell structure integrity, the accumulation of abnormal proteins under stress condition in TRP5-deleted strain, which is not capable of degrading and removing such proteins, may account for the increased sensitivity of TRP5-deleted strain to oxidative stress. These results pointed out that TRP5 plays an important role in yeast stress response. Taken together, the results suggest that both negative and positive elements are required to involve and coordinate to achieve proper and efficient response in yeast to oxidative stress.口試委員會審定書..................................... i 謝................................................ ii文摘要............................................. iii文摘要............................................. iv用名詞之縮寫與全名對照表........................... v 次................................................ vi目次............................................... ix目次............................................... x錄目次............................................. xi一章 前言.......................................... 1.1氧化逆境.......................................... 1.1.1 ROS之生合成.................................... 1.2.1 ROS之來源...................................... 2.3.1氧化逆境對酵母菌生理發育的影響.................. 3.2抗氧化防禦系統(antioxidant defense system)........ 4.2.1抗氧化酵素...................................... 4.2.1.1超氧岐化脢 (superoxide dismutase，SOD)........ 4.2.1.2過氧化酶 (catalase, CAT)...................... 4.2.1.3過氧化物酶 (peroxidase)....................... 4.2.2 非酵素型抗氧化物............................... 5.3 YGR021w (DUF28)功能相關研究...................... 7.4 YGL026c (TRP5)功能相關研究....................... 8.5研究目標.......................................... 9二章 材料與方法.................................... 10.1 YGR021w及TRP5基因選殖............................ 10.1.1 酵母菌genomic DNA萃取.......................... 10.1.2 反轉錄聚合酶連鎖反應(RT-PCR)................... 10.1.3 限制酶的消化水解............................... 11.1.4 載體與基因之接合............................... 11.1.5製備大腸桿菌勝任細胞的製備與轉型作用............ 12.1.5.1大腸桿菌勝任細胞的製備........................ 12.1.5.2大腸桿菌勝任細胞的轉型作用.................... 12.1.6酵母菌勝任細胞的製備與轉型作用.................. 12.1.6.1酵母菌勝任細胞的製備.......................... 12.1.6.2酵母菌勝任細胞的轉型作用...................... 13.2逆境處理對酵母菌的菌落形成和生長曲線之測試........ 13.2.1氧化及不同逆境處理下菌落形成之測試.............. 13.2.2 H2O2氧化逆境處理下液態培基中生長曲線之測試..... 13.3 RNA expression................................... 14.3.1 RNA製備(RNA preparation)....................... 14.3.2北方點墨法(Northern Blotting)................... 14.3.2.1 1%甲醛電泳膠體製備(1% formaldehyde gel)...... 14.3.2.2總核糖核酸轉印(Total RNA transfer)............ 14.3.2.3製作DIG標記探針 (probe)....................... 15.3.2.4雜合反應 (Hybridization)...................... 15.3.2.5免疫探知作用 (Immunological detection)........ 16.4外加胺基酸實驗.................................... 16.5微陣列(Microarray)................................ 17三章 結果.......................................... 18.1氧化逆境下酵母菌未知功能基因YGR021w (DUF28 family)之功能分析................................................. 18.1.1生物資訊 (bioinformatics)功能性研究............. 18.1.1.1 DUF28蛋白在不同物種之分類範圍(taxonomic coverage)及omologues演化樹(phylogenetic trees)分析............. 18.1.2不同氧化劑處理之氧化逆境下YGR021w RNA之表現..... 18.1.3檢測實驗所需之酵母菌菌株中YGR021w基因之表現..... 19.1.4多種逆境處理對酵母菌菌落形成及生長之影響........ 19.1.5酵母菌粒線體活性之測試.......................... 20.1.6不同氧化劑處理對酵母菌生長之影響................ 20.1.7抗氧化相關基因在WT及YGR021w△在氧化逆境下之表現 20.1.8 DNA微陣列(Microarray)分析...................... 21.1.9 DNA微陣列實驗所得基因之驗證.................... 23.2氧化逆境下酵母菌YGL026c (Tryptophan systemase, TRP5)功能分析........................................... 26.2.1不同氧化劑處理之氧化逆境下TRP5 RNA之表現........ 26.2.2檢測實驗所需之酵母菌菌株中TRP5基因之表現........ 26.2.3多種逆境處理對酵母菌菌落形成及生長率之影響...... 26.2.4抗氧化相關基因在WT及trp5△遭遇氧化逆境之表現.... 27.2.5外加不同胺基酸抵抗氧化逆境之效果................ 28.2.6缺氮飢餓 (nitrogen starvation)試驗.............. 29.2.7 DNA微陣列Microarray 分析....................... 29.2.8 DNA微陣列篩選之目標基因之驗證.................. 31四章 討論.......................................... 33.1氧化逆境下酵母菌未知功能基因YGR021w功能機制之探討. 33.1.1 YGR021w基因在不同氧化劑(oxidants)處理下之表現與探討................................................... 34.1.2 YGR021w基因缺失在不同逆境及不同碳源下之存活能力之討................................................. 35.1.3 DNA微陣列實驗中可能受YGR021w影響的基因之可能功能探討................................................... 35.1.3.1 DNA微陣列實驗之探討.......................... 35.1.3.2 YGR021w△在酵母菌遭遇氧化逆境時影響之基因群表現析................................................. 40.1.3.3 DNA微陣列實驗之驗證.......................... 40.2氧化逆境下酵母菌YGL026c (TRP5)功能機制之探討...... 43.2.1 DNA微陣列實驗中可能受TRP5影響的基因之可能功能探討................................................... 43.2.3.1 trp5△在酵母菌正常生長條件下所影響之基因群表現 43.3.2.2 trp5△在酵母菌遭遇氧化逆境時所影響之基因群表現 45五章 未來展望....................................... 48考文獻.............................................. 49目次一、使用酵母菌菌株及質體(Yeast strains and plasmid used in this work)......................................... 56二、酵母菌抗氧化微陣列中在氧化逆境下可能受YGR021w影響基因................................................ 57三、酵母菌抗氧化微陣列中在正常生長條件下可能受TRP5影響基因................................................ 62四、酵母菌抗氧化微陣列中在氧化逆境下可能受TRP5影響之基因.................................................... 66目次 一、YGR021w在氧化逆境下之表現....................... 72二、分析本論文使用之各式酵母菌菌株中YGR021w之表現... 73三、WT和YGR021w△於營養培養基上處理不同逆境及碳源的生情形................................................ 74四、WT和YGR021w△於貧瘠培養基上處理不同逆境及碳源的生情形................................................ 75五、WTc與YGR021w overexpression strain於處理H2O2下之生情形................................................ 76六、WT和YGR021w△處理H2O2，培養16小時後之生長情形... 77七、以半定量RT-RCR分析WT和YGR021w△之氧化逆境相關基因.................................................... 78八、酵母菌在氧化逆境下可能被YGR021w影響的基因群之功能配圖................................................ 79九、以即時定量RT-RCR分析已知與逆境反應相關之基因的表現.................................................... 80十、以即時定量RT-RCR分析與逆境反應相關之新穎基因的表現.................................................... 81十一、YGL026c(TRP5)在氧化逆境下之表現............... 82十二、分析本論文使用之各式酵母菌菌株中TRP5之表現.... 83十三、WT和trp5△於營養培養基上處理不同逆境的生長情形比較.................................................... 84十四、WT和trp5△於貧瘠培養基上處理不同逆境的生長情形比較.................................................... 85十五、WTc與TRP5 overexpression strain於處理不同逆境之長情形.............................................. 86十六、WT和trp5△處理H2O2，培養16小時後之生長情形比較 87十七、以半定量RT-RCR分析WT和trp5△之氧化逆境相關基因 88十八、外加tryptophan、IAA、methionine試驗........... 89十九、缺氮飢餓試驗(nitrogen starvation test)........ 90二十、酵母菌在正常生長下可能被TRP5影響的基因群之功能配圖................................................ 91二十一、酵母菌在氧化逆境下可能被TRP5影響的基因群之功分配圖.............................................. 92二十二、以即時定量RT-RCR分析微陣列篩選之基因的表現.. 93錄目次 錄一、本研究使用之引子序列.......................... 94錄二、利用生物資訊軟體InterPro分析DUF28蛋白......... 95錄三、DUF28 homologues演化樹(phylogenetic trees)分析 96錄四、酵母菌色胺菌之合成路徑........................ 97錄五、YGR021w在氧化逆境下可能參與之相關機制模式圖... 98錄六、TPR5在氧化逆境下可能參與之相關機制模式圖...... 99錄七、常用培養基配方................................100application/pdf1438838 bytesapplication/pdfen-US酵母菌氧化逆境DUF28蛋白色胺酸S. cerevisiaeoxidative stressDUF28tryptophanhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181946/1/ntu-98-R95b42032-1.pdf