本文關(guān)鍵詞： 白念珠菌 蛋白磷酸酯酶 CaPTC2 CaPPH3 CaPTC5　出處：《天津大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】：蛋白質(zhì)的可逆磷酸化是真核細(xì)胞信號傳導(dǎo)途徑中的一個(gè)關(guān)鍵調(diào)控機(jī)制,蛋白激酶和蛋白磷酸酯酶分別調(diào)控底物的磷酸化狀態(tài),從而實(shí)現(xiàn)胞內(nèi)信號途徑的激活或失活。Ser/Thr蛋白磷酸酯酶是胞內(nèi)重要的一類蛋白磷酸酯酶亞家族,其中2C類蛋白磷酸酯酶是一類特異依賴于鎂離子或錳離子的單體酶。為挖掘抗真菌藥物的作用靶點(diǎn),本文對白念珠菌CaPTC2、CaPPH3和CaPTC5基因的功能進(jìn)行了研究。 利用釀酒酵母CaPtc2p、CaPph3p和CaPtc5p的氨基酸序列在白念珠菌數(shù)據(jù)庫中進(jìn)行搜索比對,我們獲得了白念珠菌CaPTC2,CaPPH3和CaPTC5的基因序列。CaPTC2的ORF全長1749 bp,編碼583個(gè)氨基酸,CaPTC2與ScPTC2所編碼蛋白的氨基酸序列相同性為31.9%。通過對CaPtc2p的催化域進(jìn)行體外表達(dá)和蛋白磷酸酯酶活性測定,發(fā)現(xiàn)重組蛋白具有去磷酸化活性,并且這種活性依賴于Mg~(2+)或Mn~(2+)的激活,說明CaPtc2p是一種PP2C類蛋白磷酸酯酶。我們采用Ura-Blaster方法敲除了CaPTC2的兩個(gè)等位基因,發(fā)現(xiàn)該基因的缺失株對SDS、唑類抗真菌藥物以及遺傳毒性試劑HU和MMS敏感。通過熒光定位實(shí)驗(yàn)發(fā)現(xiàn)該蛋白主要定位于線粒體,同時(shí)還存在于細(xì)胞質(zhì)里。 CaPPH3基因的ORF全長783 bp,編碼261個(gè)氨基酸。白念珠菌CaPPH3與釀酒酵母ScPPH3所編碼蛋白的氨基酸序列相同性為68.2%。我們利用同樣的方法構(gòu)建了pph3△/pph3△缺失株。結(jié)果發(fā)現(xiàn),該缺失株對遺傳毒性試劑HU和MMS敏感,并且在液體菌絲誘導(dǎo)培養(yǎng)基中菌絲生成速度加快。此外,我們還發(fā)現(xiàn),CaPTC2和CaPPH3雙基因共同缺失后,雙缺失株對遺傳毒性試劑的敏感性表型呈現(xiàn)疊加的現(xiàn)象,推測他們可能通過不同的信號通路參與DNA損傷修復(fù)過程。 CaPTC5基因的ORF全長1740 bp,編碼580個(gè)氨基酸。CaPtc5p與ScPtc5p的氨基酸序列相同性為39.7%。我們構(gòu)建了ptc5△/ptc5△缺失株,但沒有發(fā)現(xiàn)缺失株有任何表型缺陷。通過熒光定位實(shí)驗(yàn)發(fā)現(xiàn)該蛋白定位于線粒體。
[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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R379

【共引文獻(xiàn)】

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1 蘭斌,劉炳亞,張濟(jì),王侃侃,陳雪華,朱正綱;胃癌細(xì)胞周期G1/S轉(zhuǎn)換期基因表達(dá)譜的分析[J];中華胃腸外科雜志;2005年03期

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2 傅文宇;從蛋白水平探討微囊藻毒素的毒作用機(jī)制[D];浙江大學(xué);2006年

3 姚利曉;日本血吸蟲絲/蘇氨酸蛋白磷酸酶新基因SjPP的研究[D];中國農(nóng)業(yè)科學(xué)院;2006年

4 邢鳴鸞;富營養(yǎng)化水體毒素對人羊膜細(xì)胞FL的毒性研究[D];浙江大學(xué);2008年

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本文編號：1542677