單一核甘酸差異來研究MIG1調控表現的機制;The Single Nucleotide Polymorphism Controlling Gene Expression of MIG1

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题名:	單一核甘酸差異來研究MIG1調控表現的機制;The Single Nucleotide Polymorphism Controlling Gene Expression of MIG1
作者:	李健銘;LI,Jian-ming
贡献者:	生命科學系
关键词:	單一核甘酸;MIG1;Single Nucleotide;Polymorphism;Gene Expression
日期:	2014-08-18
上传时间:	2014-10-15 14:39:11 (UTC+8)
出版者:	國立中央大學
摘要:	酵母菌在生長時會優先利用葡萄糖當作碳源進行發酵作用以供生長，當環境中的葡萄糖開始被消耗殆盡時，酵母菌細胞內的Mig1蛋白質會改變新陳代謝的作用，而使用其他的碳源，經由非發酵作用產生能量供自己生長。但有關於調節MIG1基因表現至今仍不清楚。先前的研究指出在MIG1基因的啟動區可能存在著4個核甘酸調控著MIG1基因的表現，這4個位置分別是-151、-152、-174和-498，其中-498的位置有可能扮演著調控MIG1基因表現的關鍵腳色位置。因此，我們藉由基因置換的方式建構-498單一核甘酸突變，來了解-498是否為調控MIG1基因的表現的唯一關鍵位置。在本次實驗的結果發現，GYL43-1在第9個小時（T9）以及GYL43-2、GYL43-3在第13個小時（T13）為diuxic shift發生的時間點，發現MIG1基因在表現量上發生了變化，然而GYL43-3的MIG1基因表現卻與其他實驗組完全相反的結果，造成原因可能是因為在調配YPAD培養基時adenine比例上的誤差導致的結果。根據焦磷酸根測序結果發現，GYL49實驗組中的gDNA（BY:RM）比值不如預期（1:1），gDNA比值呈現1:2-1:2.5，導致無法與GYL43野生型做比較。推論可能是因為在第一次基因置換時將MIG1的引子區序列改變，使得基因表現量無法達到正常水準，而在第二次基因置換時，BY實驗型將野生型RM的MIG1插入了染色體上其他的位置，而導致在GYL49實驗組的酵母菌中，RM的MIG1基因數量比BY來得高。 ;Yeasts preferentially use glucose as their carbon source via fermentation for growth. When glucose in the environment is exhausted, yeasts will adjust their metabolisms though Mig1 in order to use other carbon sources via non-fermentation processes for growth. However, the regulation of MIG1 expression remains unclear. Previous studies have indicated that four nucleotides, at positions -151, -152, -174 and -498, in the promoter region might control MIG1 expression levels, and the nucleotide located at -498 may play a key role in regulating MIG1 expressions. Therefore, we constructed a single nucleotide mutant strain by swapping to determine whether the nucleotide at position -498 is the only key regulatory. Based on our results, we found out that GYL43-1 at the ninth hour（T9） and GYL43-2、GYL43-3 at the thirteenth hour（T13） time point were diuxic shift. But the MIG1 expression of GYL43-3 was opposite to the previous study results, and we inferred that our adenine concentrations were highly variable in YPAD media. Based our pyrosequencing results, we found out that gDNA ratios （BY:RM） were not 1:1 in the experimental group, GYL49. The ratios of GYL49 gDNA were 1:2 to 1:2.5, therefore we could not compare the gene expression levels with the wild-type group, GYL43, to determine whether the nucleotide at position -498 is the only key regulatory. We inferred when we changed the promoter sequence after the first swapping, making the MIG1 expression level lower than normal, and during the second swapping, the BY strain inserted the wild-type RM strain`s MIG1 gene fragments into the chromosome at other sites. Therefore the MIG1 gene expression level of RM was higher than the one of BY in the experimental group, GYL49.
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