酿酒酵母细胞中影响蛋白激酶Rck2表达的基因筛选和分析
发布时间：2018-07-17 21:18
【摘要】：酿酒酵母细胞中的RCK2基因编码的蛋白激酶Rck2是高渗甘油途径(HOG)中的蛋白激酶Hog1在细胞质中的作用底物,其被磷酸化而激活,从而应答胞外环境的变化。为了筛选出酵母基因组中调控Rck2表达的基因,本论文将表达质粒pHAC111-RCK2-HA导入酿酒酵母的所有非必需基因缺失株中,利用Western Blot检测融合蛋白Rck2-HA的表达量。最后筛选出13个影响Rck2表达的基因。对这些基因的功能进行分析和研究,结果发现:这些基因的功能主要是与细胞壁的完整性、线粒体功能和细胞的分裂繁殖有关。这些基因主要是通过影响RCK2的转录水平来影响Rck2的表达。除了SAP190和KRE2外,其余基因的缺失都使Hog1的磷酸化水平降低,这说明它们都与HOG途径有关。此外,通过表型实验发现RTF1、KRE6、EMC5和KRE28基因的缺失导致细胞对细胞壁干扰剂敏感。同时,在镉胁迫时,MTC3、RTF1、KRE6、EMC5、KRE2和MRLP39基因的缺失使Slt2的磷酸化水平降低,说明这些基因参与镉胁迫诱导的CWI途径的激活。另外对负调控细胞壁完整性途径(CWI)途径的4个磷酸酯酶基因PTP2、PTP3、SDP1和MSG5的研究发现,PTP2和MSG5的缺失也会导致细胞对细胞壁干扰剂敏感;而在镉诱导下,MSG5基因的缺失,使Slt2的磷酸化水平升高,使细胞对镉有一定的耐受性,所以Msg5是负调控CWI途径中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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ925
，
本文编号：2130965
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ925
，
本文编号：2130965
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