調控酵母菌MIG1基因表達的轉錄因子｜國立中央大學博碩士論文系統

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研究生：	鄭又銘 Yu-ming Cheng
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
指導教授：	劉阜果 Fu-guo Liu
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2015
畢業學年度：	103
語文別：	中文
論文頁數：	57
中文關鍵詞：	酵母菌、一次雜交
外文關鍵詞：	one-hybrid, MIG1
相關次數：	點閱：14 下載：0
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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.

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

 

                                

Carlson, M., Osmond, B. C., Neigeborm, L., Bostein, D., (1984) A suppressor of snf1 mutations causes constitutive high-level invertase synthesis in yeast. Genetics 107: 19-32.

Carlson, M., (1999) Glucose repression in yeast. Microbiology 2: 202-207.

Chang, Y. W., Liu, F. G., Yu, N., Sung, H. M., (2008) Roles of cis- and trans-Changes in the Regulatory Evolution of Genes in the gluconeogenic Pathway in Yeast. Mol. Biol. 25: 1863–1875.

Dikicioglu, D., Pir, P., Oliver, S. G., (2013) Predicting complex phenotype–genotype interactions to enable yeast engineering: Saccharomyces cerevisiae as a model organism and a cell factory. Biotechol. J. 8: 1017-1034.

Goffeau, A., Barrell, B. G., Bussey, H., Davis, R. W., Dujon, B., Feldmann, H., Galibert, F., Hoheisel, J. D., Jacq, C., Johnston, M., Louis, E. J., Mewes, H. W., Murakami, Y., Philippsen, P., Tettelin, H., Oliver, S. G., (1996) Life with 6000 Genes. Science 274: 546 563–567.

Hahn, S., Young, E. T., (2011) Transcriptional Regulation in Saccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators. Genetics 189: 705–736.

Johnston, M., Carlson, M., (1992) Regulation of carbon and phosphate utilization. In: Jones E. W., Pringle, J. R., Broach, J. R., editors. The molecular and cellular biology of the yeast Saccharomyces gene expression. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. p.193–281.

Klein, C. J., Olsson, L., Rønnow, B., Mikkelsen, J. D., Nielsen, J., (1996) Alleviation of glucose repression of maltose metabolism by MIG1 disruption in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 62: 4441–4449.

Kurtzman, C. P., Fell, J. W., (2006) Yeast Systematics and Phylogeny—Implications of Molecular Identification Methods for Studies in Ecology. The Yeast Handbook, Germany: Springer-Verlag Berlin Herdelberg. p. 11-30.

Legras, J. L., Merdinoglu, D., Cornuet, J. M., Karst, F., (2007) Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history. Mol. Ecol. 16: 2091-2102.

Lemon, B., Tjian, R., (2000) Orchestrated response: a symphony of transcription factors for gene control. Genes. Dev. 14: 2551-2569.

Lundin, M., Nehlin, J. O., Ronne, H., (1994) Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein Mig1. Mol. Cell. Biol. 14: 1979-1985

Nehlin, J. O., Ronne, H., (1990) Yeast Mig1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. EMBO. 9: 2891-2898.

Ostling, J., Ronne, H., (1998) Negative control of the Mig1p repressor by Snf1p-dependent phosphorylation in the absence of glucose. Eur. J. Biochem. 252: 162–168.

Ouwerkerk, P. B., Meijer, A. H., (2011) Yeast one-hybrid screens for detection of transcription factor DNA interactions. Methods. Mol. Biol. 678: 211-227.

Prescott, L. M., Harley, J. P., Klein, D. A., (2002) Microbiology, 5th ed. McGraw-Hill, Boston, p. 554.

Suh, S. O., McHugh, J. V., Pollock, D. D., Blackwell, M., (2005) The beetle gut: a hyperdiverse source of novel yeasts. Mycol. Res. 109: 261–265.

Schuller, H. J., (2003) Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae. Curr. Genet. 43: 139–160.

Takesako, K., Kuroda, H., Inoue, T., Haruna, F., Yoshikawa, Y., Kato, I., Uchida, K., Hiratani, T., Yamaguchi, H., (1993) Biological properties of aureobasidin A, a cyclic depsipeptide antifungal antibiotic. J. Antibiot. 46: 1414-1420.

Teste, M. A., Duquenne, M., François, J. M., Parrou, J. L., (2009) Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC. Mol. Biol. 10: 99.

Turcotte, B., Liang, X. B., Robert, F., Soontorngun, N., (2010) Transcriptional regulation of nonfermentable carbon utilization in budding yeast. FEMS. Yeast. Res. 10: 2-13.

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