調控酵母菌MIG1基因表達的轉錄因子;Yeast one-hybrid to determine transcription factors of MIG1

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题名:	調控酵母菌MIG1基因表達的轉錄因子;Yeast one-hybrid to determine transcription factors of MIG1
作者:	鄭又銘;Cheng,Yu-ming
贡献者:	生命科學系
关键词:	酵母菌;一次雜交;yeast;one-hybrid;MIG1
日期:	2015-01-13
上传时间:	2015-03-16 15:12:03 (UTC+8)
出版者:	國立中央大學
摘要:	MIG1基因在酵母菌Saccharomyces cerevisiae中，對於細胞內葡萄糖的代謝扮演一個重要的角色。然而， MIG1基因如何被調控還不明確。因此，在我的研究中，我想找出MIG1基因在轉錄過程中是如何被調控的。從先前的研究可得知MIG1基因啟動子上的-498、-174、-152、-151是影響BY和RM/YJM strain在葡萄糖缺乏時基因表現有差異的重要位置。因此，我利用Matchmaker® Gold Yeast One-Hybrid Library Screening System (Clontech)來找出哪一個轉錄因子會結合到啟動子的這些位置上來調節MIG1基因表現。我將MIG1啟動子-498到-151區間建構到pAbA載體上，然後建構出一個含有AbAr 報導基因的酵母菌種Y1H Gold[pAbA-MIG1]。接著我將pGADT7-rec載體和酵母菌cDNA library轉殖到Y1H Gold[pAbA-MIG1]裡，並將酵母菌培養在SD/-Leu/AbA200培養基中。我挑選了生長的菌落並做基因定序，並利用Saccharomyces Genome Database而查出這些基因YPL077C、YFR031C-A、YAL039C、YOR285W、YER081W、YML063W、YJR105W、YDR152W、YLR325C、YBR047W、YDL083C、YBR015C和YAL012W。我利用這些基因的knock out strain藉由RT-PCR來看MIG1的基因表現，結果我發現這些基因並不會影響MIG1。接著我將包含-498到-151的MIG1啟動子序列利用網路上的Transcription factors prediction推測出YDR216W和YFR034C會結合在這區間上。但在做完RT-PCR後，這兩個基因也不會影響 MIG1基因表現。因此，未來我們可能會試著先找出培養基中葡萄糖被耗盡的時間點，在那個時間點之後再收菌，這樣或許MIG1基因在knock out strain和wild-type之間的基因表現量就會有差異，進而找出是哪一個蛋白質在調控MIG1。 ;MIG1 is important to regular glucose metabolism in yeast, Saccharomyces cerevisiae. However it’s not clear how MIG1 gene expression is regulated. Therefore, in my research I’m interested in finding out regulation of MIG1 transcription. Based on previous research, we have already known at MIG1 promoter region -498, -174, -152 and -151 are important sites to cause gene expression differences between BY and RM/YJM strains on glucose depletion. Therefore, I used Matchmaker® Gold Yeast One-Hybrid Library Screening System (Clontech) to detect which transcription factors bind the promoter region to regulate MIG1 expression. I constructed MIG1 promoter region into pAbA vector and created a AbAr reporter yeast strain Y1H Gold[pAbA-MIG1]. Then, I transformed pGADT7-rec with yeast cDNA library into Y1H Gold[pAbA-MIG1] strain and cultured the cell on the SD/-Leu/AbA200 agar plates. I picked the colonies for sequencing and got these genes YPL077C、YFR031C-A、YAL039C、YOR285W、YER081W、YML063W、YJR105W、YDR152W、YLR325C、YBR047W、YDL083C、YBR015C and YAL012W by Saccharomyces Genome Database. I used those gene-knock-out strains to detect MIG1 gene expression by doing RT-PCR and found those genes didn’t affect MIG1 gene expression. Then, I used the sequence of MIG1 promoter region including -151~ -498 to do transcription factors prediction and found out YDR216W and YFR034C bind on this region. But after doing RT-PCR, they also didn’t affect MIG1 gene expression. Therefore, maybe we will detect the time that glucose is depleted in the medium then we stop the cell growth on that time. Perhaps MIG1 gene expression between knock strains and wild-type will be different, and we can find out which transcription factors regulate MIG1 gene expression.
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