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標題: 酵母菌耐高溫之實驗演化
Experimental Evolution of Yeast for High Temperature Tolerance
作者: 黃志仁
Chih-Jen Huang
關鍵字: 耐熱;逆境適應;實驗演化學;集群分離分析法;數量性狀基因座;thermotolerance;stress adaptation;experimental evolution;high temperature growth;protein complexes;pooled segregant analysis
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'耐熱'是一種由多基因決定的性狀,用以描述在高溫的環境下,生物體的存活的能力與生長的情形。即便有零星的耐熱相關基因被發現,生物如何逐漸累積自身的突變以適應高溫的環境卻還不是那麼的清楚。在本研究中,我利用實驗演化學的方法以階梯式的漸進升溫,成功地將酵母菌的耐熱生長溫度提高到了42度。利用全基因體定序技術,我解序了14株親本及序列演化品系,並從其中發掘出153個不同的突變,囊括了各種點突變、小插入缺失以及長片段的染色體區段性複製/缺失變異。這些突變發生之後,有些突變隨溫度上升至42度的演化過程中被保留下來,某種程度上代表了這些突變與耐熱生長的性狀有所關聯。利用功能性分類工具,我發現到一些突變傾向於發生在某些蛋白質複合體上,例如SWI/SNF 複合體以及F型三磷酸腺苷合成酶。同時,也看到一些突變發生在逆境調控相關的訊息傳遞路徑上,例如Hog1, RAS-cAMP, 以及 Rho1-Pkc 等。這些研究結果顯示,要達到耐熱生長,部分可藉由修改現有的逆境調控機制來達成。利用集群分離分析法,我進一步分析了五組不同耐熱程度的分離子樣本群,找到了42度耐熱的關鍵基因突變組,並進一步定義了其中較具高顯性的突變。利用分子遺傳學方法,也成功的在親本的遺傳背景下,以功能性實驗驗證了這些突變所帶來的耐熱效果。本研究顯著地拓展了我們對酵母菌耐熱遺傳基礎的了解。

Thermotolerance is a polygenic trait that contributes to cell survival and growth under unusually high temperatures. Although some genes associated with high temperature growth (Htg+) have been identified, how cells accumulate mutations to achieve prolonged thermotolerance is still mysterious. Here I conducted experimental evolution of a Saccharomyces cerevisiae laboratory strain with stepwise temperature increases for it to grow at 42 °C. Whole genome resequencing of 14 evolved strains and the parental strain revealed a total of 153 mutations in the evolved strains, including single nucleotide variants, small INDELs, and segmental duplication/deletion events. Some mutations persisted from an intermediate temperature to 42 °C, so they might be Htg+ mutations. Functional categorization of mutations revealed enrichment of exonic mutations in the SWI/SNF complex and F-type ATPase, pointing to their involvement in high temperature tolerance. In addition, multiple mutations were found in a general stress associated signal transduction network consisting of Hog1 mediated pathway, RAS-cAMP pathway, and Rho1-Pkc1 mediated cell wall integrity pathway, implying that cells can achieve Htg+ partly through modifying existing stress regulatory mechanisms. Using pooled segregant analysis of 5 Htg+ phenotype-orientated pools, I inferred causative mutations for growth at 42 °C and identified those mutations with stronger impacts on the phenotype. Finally, I experimentally validated a number of the candidate Htg+ mutations. This study increased our understanding of the genetic basis of yeast tolerance to high temperature.
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