本文關(guān)鍵詞：白色念珠菌基因CaTCO89和CaPTC1的鑒定及功能研究　出處：《天津大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 白色念珠菌 釀酒酵母 CaTCO89 CaPTC1 TOR途徑 HOG途徑 互補(bǔ)試驗(yàn)

【摘要】： 白色念珠菌是臨床上一種最重要的條件致病真菌。目前,人們對(duì)白色念珠菌的研究主要集中在弄清其致病機(jī)理和發(fā)現(xiàn)新的藥物作用靶點(diǎn)。通過(guò)用釀酒酵母基因編碼的氨基酸序列對(duì)斯坦福大學(xué)白色念珠菌基因組數(shù)據(jù)庫(kù)進(jìn)行比對(duì),我們得到白色念珠菌基因CaTCO89和CaPTC1的序列。基因CaTCO89的ORF長(zhǎng)為2127 bp,編碼708個(gè)氨基酸。CaTCO89與釀酒酵母ScTCO89所編碼蛋白氨基酸序列的相似性為27.5%。我們發(fā)現(xiàn)CaTCO89基因可以互補(bǔ)ScTCO89在雷帕霉素和氯化鋰抗性以及保持細(xì)胞壁完整性方面的功能。我們以白色念珠菌菌株RM1000為背景,構(gòu)建了CaTCO89的雙缺失菌株TJU4(tco89/tco89)。與RM1000相比,TJU4對(duì)雷帕霉素和氯化鋰的敏感性增加。在TJU4細(xì)胞中異位表達(dá)CaTCO89基因能夠恢復(fù)TJU4對(duì)雷帕霉素抗性到野生型水平,然而卻顯著增強(qiáng)TJU4細(xì)胞對(duì)氯化鋰和過(guò)氧化氫的抗性以及對(duì)Cd2+的敏感性。此外,在Spider和SD-Ura液體培養(yǎng)基中,CaTCO89基因在TJU4中的異位表達(dá)能引起細(xì)胞凝集形成絮狀沉淀。在Spider固體培養(yǎng)基上,TJU4的菌落形態(tài)與野生型菌株相同,為邊緣不整齊扁平狀、表面凸凹不平,且有周邊菌絲形成,但是異位表達(dá)CaTCO89的TJU4菌落形態(tài)表現(xiàn)為表面光滑的面包狀。 基因CaPTC1的ORF長(zhǎng)度為1128 bp,編碼375個(gè)氨基酸,與釀酒酵母ScPtc1p的相似性為52%。CaPTC1的雙缺失菌株SYY4(ptc1/ptc1)對(duì)Li+、Na+和K+離子敏感,異位表達(dá)CaPTC1能夠抑制SYY4細(xì)胞的缺失表型。我們克隆并在細(xì)菌中表達(dá)了CaPtc1p的催化區(qū)域,發(fā)現(xiàn)純化的重組蛋白具有去磷酸化活性,這種活性還依賴于Mn2+或Mg2+的存在,并且受到絲氨酸-蘇氨酸去磷酸酯酶抑制劑NaF的抑制。這些結(jié)果表明CaPtc1p是一種PP2C類蛋白磷酸酯酶。
[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.

【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R379

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相關(guān)期刊論文 前1條

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本文編號(hào)：1400441