Yeast one-hybrid to determine transcription factors of MIG1

• Main Authors: Yu-ming Cheng, 鄭又銘
• Other Authors: Fu-guo Liu
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/58784153392029127180

碩士 === 國立中央大學 === 生命科學系 === 103 === 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.