白色念珠菌基因CaTCO89和CaPTC1的鉴定及功能研究

发布时间：2018-01-09 07:08

  本文关键词：白色念珠菌基因CaTCO89和CaPTC1的鉴定及功能研究　出处：《天津大学》2007年博士论文　论文类型：学位论文

  更多相关文章： 白色念珠菌 酿酒酵母 CaTCO89 CaPTC1 TOR途径 HOG途径 互补试验

【摘要】： 白色念珠菌是临床上一种最重要的条件致病真菌。目前,人们对白色念珠菌的研究主要集中在弄清其致病机理和发现新的药物作用靶点。通过用酿酒酵母基因编码的氨基酸序列对斯坦福大学白色念珠菌基因组数据库进行比对,我们得到白色念珠菌基因CaTCO89和CaPTC1的序列。基因CaTCO89的ORF长为2127 bp,编码708个氨基酸。CaTCO89与酿酒酵母ScTCO89所编码蛋白氨基酸序列的相似性为27.5%。我们发现CaTCO89基因可以互补ScTCO89在雷帕霉素和氯化锂抗性以及保持细胞壁完整性方面的功能。我们以白色念珠菌菌株RM1000为背景,构建了CaTCO89的双缺失菌株TJU4(tco89/tco89)。与RM1000相比,TJU4对雷帕霉素和氯化锂的敏感性增加。在TJU4细胞中异位表达CaTCO89基因能够恢复TJU4对雷帕霉素抗性到野生型水平,然而却显著增强TJU4细胞对氯化锂和过氧化氢的抗性以及对Cd2+的敏感性。此外,在Spider和SD-Ura液体培养基中,CaTCO89基因在TJU4中的异位表达能引起细胞凝集形成絮状沉淀。在Spider固体培养基上,TJU4的菌落形态与野生型菌株相同,为边缘不整齐扁平状、表面凸凹不平,且有周边菌丝形成,但是异位表达CaTCO89的TJU4菌落形态表现为表面光滑的面包状。 基因CaPTC1的ORF长度为1128 bp,编码375个氨基酸,与酿酒酵母ScPtc1p的相似性为52%。CaPTC1的双缺失菌株SYY4(ptc1/ptc1)对Li+、Na+和K+离子敏感,异位表达CaPTC1能够抑制SYY4细胞的缺失表型。我们克隆并在细菌中表达了CaPtc1p的催化区域,发现纯化的重组蛋白具有去磷酸化活性,这种活性还依赖于Mn2+或Mg2+的存在,并且受到丝氨酸-苏氨酸去磷酸酯酶抑制剂NaF的抑制。这些结果表明CaPtc1p是一种PP2C类蛋白磷酸酯酶。
[Abstract]:Candida albicans is clinically the most important fungal pathogen. At present, studies on C. albicans have been focused on understanding its pathogenesis and find new drug targets. The amino acid sequence of yeast gene encoding of Stanford University Candida albicans genome database for comparison, we get a sequence of Candida albicans gene the CaTCO89 and CaPTC1. CaTCO89 gene ORF length was 2127 BP, encoding 708 amino acid.CaTCO89 and Saccharomyces cerevisiae ScTCO89 encoding protein amino acid sequence similarity to 27.5%. we found that CaTCO89 could complement ScTCO89 in rapamycin and lithium chloride resistance and maintain cell wall integrity function. We Candida albicans strain RM1000 as the background and build a CaTCO89 double mutant strain TJU4 (tco89/tco89). Compared with RM1000, TJU4 of rapamycin and chloride The increase of lithium sensitivity in TJU4 cells. Ectopic expression of CaTCO89 gene can restore TJU4 to rapamycin resistance to the level of the wild type, but significantly increased the resistance of TJU4 cells to lithium chloride and hydrogen peroxide and sensitivity to Cd2+. In addition, in the medium of Spider and SD-Ura in the liquid, ectopic expression of CaTCO89 gene in TJU4 can cause cell agglutination formation of floc. In Spider solid medium, colony morphology and wild type strain TJU4 the same as irregular edge flat, uneven surface, and the surrounding hyphal formation, but the ectopic expression of CaTCO89 TJU4 colony form is the smooth surface of the bread.
CaPTC1 gene ORF was 1128 BP in length, encoding 375 amino acid similarity with Saccharomyces cerevisiae ScPtc1p 52%.CaPTC1 double mutant strain SYY4 (ptc1/ptc1) of Li+, Na+ and K+ ion sensitive, ectopic expression of CaPTC1 can inhibit SYY4 cell deletion phenotype. We cloned and expressed in bacteria in the catalytic region of CaPtc1p and found that the purified recombinant protein with the dephosphorylation activity, this activity also depends on Mn2+ or Mg2+, and by the inhibition of serine threonine phosphatase inhibitor to NaF. These results suggest that CaPtc1p is a PP2C protein phosphatase.

【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R379

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