【摘要】：釀酒酵母細胞中的RCK2基因編碼的蛋白激酶Rck2是高滲甘油途徑(HOG)中的蛋白激酶Hog1在細胞質(zhì)中的作用底物,其被磷酸化而激活,從而應答胞外環(huán)境的變化。為了篩選出酵母基因組中調(diào)控Rck2表達的基因,本論文將表達質(zhì)粒pHAC111-RCK2-HA導入釀酒酵母的所有非必需基因缺失株中,利用Western Blot檢測融合蛋白Rck2-HA的表達量。最后篩選出13個影響Rck2表達的基因。對這些基因的功能進行分析和研究,結(jié)果發(fā)現(xiàn):這些基因的功能主要是與細胞壁的完整性、線粒體功能和細胞的分裂繁殖有關。這些基因主要是通過影響RCK2的轉(zhuǎn)錄水平來影響Rck2的表達。除了SAP190和KRE2外,其余基因的缺失都使Hog1的磷酸化水平降低,這說明它們都與HOG途徑有關。此外,通過表型實驗發(fā)現(xiàn)RTF1、KRE6、EMC5和KRE28基因的缺失導致細胞對細胞壁干擾劑敏感。同時,在鎘脅迫時,MTC3、RTF1、KRE6、EMC5、KRE2和MRLP39基因的缺失使Slt2的磷酸化水平降低,說明這些基因參與鎘脅迫誘導的CWI途徑的激活。另外對負調(diào)控細胞壁完整性途徑(CWI)途徑的4個磷酸酯酶基因PTP2、PTP3、SDP1和MSG5的研究發(fā)現(xiàn),PTP2和MSG5的缺失也會導致細胞對細胞壁干擾劑敏感;而在鎘誘導下,MSG5基因的缺失,使Slt2的磷酸化水平升高,使細胞對鎘有一定的耐受性,所以Msg5是負調(diào)控CWI途徑中Slt2的主要蛋白磷酸酯酶。
[Abstract]:The protein kinase Rck2 encoded by RCK2 gene in Saccharomyces cerevisiae cells is the substrate of protein kinase Hog1 in the hypertonic glycerol pathway (HOG), which is activated by phosphorylation to respond to changes in the extracellular environment. In order to screen the genes regulating the expression of Rck2 in yeast genome, the expression plasmid pHAC111-RCK2-HA was introduced into Saccharomyces cerevisiae to detect the expression of Rck2-HA by Western Blot. Finally, 13 genes affecting Rck2 expression were screened. The function of these genes was analyzed and studied. The results showed that the functions of these genes were mainly related to the integrity of cell wall, mitochondrial function and cell division and reproduction. These genes affect the expression of Rck2 mainly by affecting the transcription level of RCK2. With the exception of SAP190 and KRE2, the loss of all the genes reduced the phosphorylation level of Hog1, suggesting that they were all related to the HOG pathway. In addition, the deletion of EMC5 and KRE28 genes resulted in cell sensitivity to cell wall interference. At the same time, the deletion of EMC5KRE2 and MRLP39 genes decreased the phosphorylation level of Slt2 during cadmium stress, suggesting that these genes are involved in the activation of CWI pathway induced by cadmium stress. In addition, the study of four phosphatase genes PTP2p3PTP3P SDP1 and MSG5 that negatively regulated the cell wall integrity pathway (CWI) showed that the deletion of PTP2 and MSG5 also led to cell wall interference sensitivity, while the deletion of MSG5 gene was induced by cadmium. The phosphorylation level of Slt2 was increased and the cells were tolerant to cadmium, so Msg5 was the main protein phosphatase that negatively regulated Slt2 in CWI pathway.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：TQ925
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本文編號：2130965