白念珠菌CaPTC2，CaPPH3和CaPTC5基因的鉴定和功能研究

发布时间：2018-02-27 12:31

本文关键词： 白念珠菌蛋白磷酸酯酶 CaPTC2 CaPPH3 CaPTC5　出处：《天津大学》2010年博士论文　论文类型：学位论文

【摘要】：蛋白质的可逆磷酸化是真核细胞信号传导途径中的一个关键调控机制,蛋白激酶和蛋白磷酸酯酶分别调控底物的磷酸化状态,从而实现胞内信号途径的激活或失活。Ser/Thr蛋白磷酸酯酶是胞内重要的一类蛋白磷酸酯酶亚家族,其中2C类蛋白磷酸酯酶是一类特异依赖于镁离子或锰离子的单体酶。为挖掘抗真菌药物的作用靶点,本文对白念珠菌CaPTC2、CaPPH3和CaPTC5基因的功能进行了研究。利用酿酒酵母CaPtc2p、CaPph3p和CaPtc5p的氨基酸序列在白念珠菌数据库中进行搜索比对,我们获得了白念珠菌CaPTC2,CaPPH3和CaPTC5的基因序列。CaPTC2的ORF全长1749 bp,编码583个氨基酸,CaPTC2与ScPTC2所编码蛋白的氨基酸序列相同性为31.9%。通过对CaPtc2p的催化域进行体外表达和蛋白磷酸酯酶活性测定,发现重组蛋白具有去磷酸化活性,并且这种活性依赖于Mg~(2+)或Mn~(2+)的激活,说明CaPtc2p是一种PP2C类蛋白磷酸酯酶。我们采用Ura-Blaster方法敲除了CaPTC2的两个等位基因,发现该基因的缺失株对SDS、唑类抗真菌药物以及遗传毒性试剂HU和MMS敏感。通过荧光定位实验发现该蛋白主要定位于线粒体,同时还存在于细胞质里。 CaPPH3基因的ORF全长783 bp,编码261个氨基酸。白念珠菌CaPPH3与酿酒酵母ScPPH3所编码蛋白的氨基酸序列相同性为68.2%。我们利用同样的方法构建了pph3△/pph3△缺失株。结果发现,该缺失株对遗传毒性试剂HU和MMS敏感,并且在液体菌丝诱导培养基中菌丝生成速度加快。此外,我们还发现,CaPTC2和CaPPH3双基因共同缺失后,双缺失株对遗传毒性试剂的敏感性表型呈现叠加的现象,推测他们可能通过不同的信号通路参与DNA损伤修复过程。 CaPTC5基因的ORF全长1740 bp,编码580个氨基酸。CaPtc5p与ScPtc5p的氨基酸序列相同性为39.7%。我们构建了ptc5△/ptc5△缺失株,但没有发现缺失株有任何表型缺陷。通过荧光定位实验发现该蛋白定位于线粒体。
[Abstract]:Protein reversible phosphorylation is a key regulation mechanism in eukaryotic signal transduction pathway. Protein kinase and protein phosphatase respectively regulate the phosphorylation of substrate. Thus, the activation or inactivation of intracellular signaling pathway. Serr / Thr protein phosphatase is an important subfamily of intracellular protein phosphatase. Among them, 2C protein phosphatase is a kind of monomeric enzyme which is specific to magnesium or manganese ions. In order to explore the target of antifungal drugs, the function of CaPTC2C2CaPPH3 and CaPTC5 genes in Candida albicans was studied in this paper. The amino acid sequences of CaPtc2pPP3p and CaPtc5p in Saccharomyces cerevisiae were searched and compared in Candida albicans database. We obtained the gene sequence of CaPTC2C2CaPPH3 and CaPTC5. The total length of ORF of CaPTC2 was 1749bp.The amino acid sequence of CaPTC2 encoding 583 amino acids was the same as that of the protein encoded by ScPTC2. The protein phosphatase activity was determined by in vitro expression of the catalytic domain of CaPtc2p. It was found that the recombinant protein has dephosphorylation activity, and this activity is dependent on the activation of Mg~(2) or Mn~(2), indicating that CaPtc2p is a kind of PP2C protein phosphatase. We use Ura-Blaster method to knock out two alleles of CaPTC2. It was found that the missing strain of the gene was sensitive to SDS, antifungal agents such as azolium and genotoxic reagents Hu and MMS. It was found by fluorescence localization that the protein was mainly located in mitochondria and also in cytoplasm. The ORF of CaPPH3 gene is 783 BP, encoding 261 amino acids. The amino acid sequence of the protein encoded by Candida albicans CaPPH3 and Saccharomyces cerevisiae ScPPH3 is 68.2%. We constructed the pph3 / pph3 deletion strain using the same method. The deletion strain was sensitive to the genotoxic reagents Hu and MMS, and the hyphal formation rate was accelerated in liquid hyphal induction medium. In addition, we also found that both CaPTC2 and CaPPH3 genes were deleted together. The susceptibility phenotypes of double deletions to genotoxic reagents were superimposed, suggesting that they might be involved in the process of DNA damage repair through different signal pathways. The ORF of CaPTC5 gene is 1740 BP, encoding 580 amino acids. CaPtc5p has the same amino acid sequence as ScPtc5p. We constructed the ptc5 / ptc5 deletion strain. However, no phenotypic defects were found in the deletion strain, which was found to be located in mitochondria by fluorescence localization assay.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R379

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