調控酵母菌MIG1基因表達的轉錄因子

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姓名

鄭又銘(Yu-ming Cheng) 查詢紙本館藏

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生命科學系

論文名稱

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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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 酵母菌
★ 一次雜交

關鍵字(英)

★ yeast
★ one-hybrid
★ MIG1

論文目次

目錄
摘要 i
Abstract ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 x
第一章緒論 1
1.1 酵母菌 1
1.1.1 酵母菌簡介 1
1.1.2 Saccharomyces cerevisiae (麵包酵母) 1
1.2 Multicopy Inhibitor of Galactose1 (MIG1)基因 2
1.2.1 MIG1 簡介1 2
1.2.2 Mig1p作用 2
1.3 Yeast one-hybrid system 3
1.3.1 Yeast one-hybrid簡介 3
1.3.2 Matchmaker® Gold Yeast One-Hybrid Library Screening System 4
1.3.3 SMART技術簡介 5
1.3.4 Aureobasidin A 5
1.4 研究目的 6
第二章材料與方法 7
2.1 酵母菌genomic DNA (gDNA)萃取 7
2.2 MIG1啟動子片段建構 7
2.3 大腸桿菌勝任細胞 (competent cells)的製備 9
2.4 大腸桿菌勝任細胞的轉殖作用 (transformation) 9
2.5 質體萃取 (plasmid extraction) 9
2.6 質體的確認 10
2.7 酵母菌勝任細胞的製備 10
2.8 酵母菌勝任細胞的轉殖作用 11
2.9 轉殖株對於Aureobasidin A最小抑制濃度的測定 12
2.10 RNA 萃取 13
2.11 First Strand SMART cDNA合成 13
2.12 藉由Long Distance PCR (LD-PCR)來放大SMART cDNA 14
2.13 利用CHROMA SPIN+TE-400 Columns來純化ds cDNA 14
2.14 菌落PCR (Colony PCR) 15
2.15 反轉錄聚合酶連鎖反應 (RT-PCR) 15
第三章結果 17
3.1 AbAr酵母菌物種的建構 17
3.2 轉殖株對於Aureobasidin A最小抑制濃度的測定 17
3.3 酵母菌cDNA library的建構 18
3.4 Y1H Gold[pAbA-MIG1]和cDNA library的one-hybrid 18
3.5 轉殖株的基因定序 (Sequencing) 19
3.6 Gene-knock-out strains的MIG1基因表現 19
第四章討論 20
參考文獻 23
圖表 26
附錄 41

圖目錄
圖 1、pAbA vector 26
圖 2、pGADT7-rec vector 27
圖 3、Create your bait reporter strain by homologous
recombination into the genome of Y1H Gold 28
圖 4、Screening for protein-DNA interactions with the Matchmaker Gold One-Hybrid System 29
圖 5、SMART cDNA synthesis generates cDNA ends that are homologous to the cloning site in pGADT7-rec 30
圖 6、Use SMART Technology and yeast biology to construct and screen your library 31
圖 7、p53-AbA vector 31
圖 8、CHROMA SPIN+TE-400 columns and collection tubes 32
圖 9、MIG1 啟動子片段 32
圖 10、利用KpnI和SalI限制酵素處理pAbA載體和MIG1啟動子
片段 33
圖 11、pAbA-MIG1轉殖 (transform)進入大腸桿菌中之
菌落PCR結果 34
圖 12、利用KpnI/SalI和NheI/SalI處理pAbA-MIG1 34
圖 13、利用BstBI處理pAbA-MIG1和p53-AbA 34
圖 14、p53-AbA和pAbA-MIG1轉殖進酵母菌Y1H Gold 35
圖 15、Y1H Gold[p53-AbA]控制組生長在不同Aureobasidin A濃度 SD/-Ura的情形 35
圖 16、Y1H Gold[pAbA-MIG1]生長在不同Aureobasidin A濃度 SD/-Ura的情形 36
圖 17、將control Mouse Liver Poly A+RNA和萃取的GYL2 RNA
利用First Strand SMART cDNA Synthesis並經由LD-PCR
放大 36
圖 18、cDNA library轉殖進Y1H Gold[pAbA-MIG1] 37
圖 19、cDNA library轉殖進Y1H Gold[pAbA-MIG1]的colony PCR
結果 37
圖 20、酵母菌gene-knock-out strains中MIG1的基因表現 (YPL077C、 YFR031C、YAL039C、YOR285W) 38
圖 21、酵母菌gene-knock-out strains中MIG1的基因表現 (YER081W、YML063W、YJR105W) 38
圖 22、酵母菌gene-knock-out strains中MIG1的基因表現 (YLR325C、YBR015C、YDL083C、YAL012W、YBR047W、
YDR152W) 39
圖 23、酵母菌gene-knock-out strains中MIG1的基因表現 (YDR216W、YFR034C) 39

表目錄

表 1、引子 (primers) 40
表 2、Y1H Gold[p53-AbA]對於Aureobasidin A濃度的表現結果 40

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指導教授

劉阜果(Fu-guo Liu)

審核日期

2015-1-13

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