本文關(guān)鍵詞： 霍亂弧菌 膽鹽 有氧生長 細(xì)胞色素c氧化酶 C-signal　出處：《南京農(nóng)業(yè)大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：霍亂是一種古老且流行廣泛的烈性腸道感染疾病,它是由革蘭氏陰性細(xì)菌霍亂弧菌所引起的。大量的研究表明,霍亂弧菌的致病能力主要是由霍亂毒素和毒素共調(diào)節(jié)菌毛引起的。 膽鹽是膽汁的一部分,在人體內(nèi)能夠幫助消化脂肪。它也是一種去垢劑,能破壞細(xì)胞膜從而殺死細(xì)胞�；魜y弧菌能夠耐受一定濃度的膽鹽,說明它有某些基因能夠把膽鹽外排出細(xì)胞或是降解膽鹽。本實驗采用轉(zhuǎn)座子隨機(jī)插入基因組的方法篩選到一株對粗膽鹽、膽酸鈉和脫氧膽酸鈉耐受能力顯著下降的霍亂弧菌突變株,經(jīng)基因測序分析發(fā)現(xiàn)此基因與文獻(xiàn)報道的霍亂弧菌膽鹽外排泵一致。說明這種利用轉(zhuǎn)座子隨機(jī)插入失活來篩選膽鹽外排相關(guān)基因的方法的建立是可行的,可以通過此體系篩選其他可能存在的膽鹽外排相關(guān)基因。 細(xì)胞色素c氧化酶是電子傳遞鏈終端氧化酶,它從細(xì)胞色素c得到電子,并把氧氣還原成水。銅離子遞呈基因scol編碼的Sc01蛋白在酵母、枯草芽孢桿菌等微生物中起到了把銅離子傳遞給細(xì)胞色素c氧化酶的銅離子氧化還原中心的作用。在霍亂弧菌中vca0038為假設(shè)的scol基因。本實通過框內(nèi)敲除的方法獲得該基因的缺失突變株后,霍亂弧菌突變株在LB液體培養(yǎng)基和M9液體培養(yǎng)基中有氧條件下生長速度并未下降,細(xì)胞色素c氧化酶氧化能力和對過氧化氫的耐受能力也沒有下降。因此,推測該基因?qū)魜y弧菌的細(xì)胞色素c氧化酶氧化能力沒有影響。 霍亂弧菌有三套群體感應(yīng)系統(tǒng)：CqsA/S系統(tǒng)、LuxS/PQ系統(tǒng)和VarS/VarA-CsrA/BCD系統(tǒng)。它們對霍亂弧菌的毒力基因表達(dá)等調(diào)控作用。群體感應(yīng)信號分子C-Signal在粘細(xì)菌Myxococcus xanthus有氧生長時細(xì)胞聚集并形成孢子的過程中起作用。這提示在霍亂弧菌中,假設(shè)的C-signal合成基因vc1815的產(chǎn)物對霍亂弧菌的群體感應(yīng)系統(tǒng)可能有所影響。本實驗缺失該基因后,霍亂弧菌突變株的生物膜形成能力并未受明顯影響,其毒力共調(diào)節(jié)菌毛TCP的合成能力也未下降,在乳鼠腸道內(nèi)的定殖能力也未受到影響,推測該基因?qū)魜y弧菌群體感應(yīng)系統(tǒng)可能沒有影響。 本實驗通過轉(zhuǎn)座子插入失活的方式構(gòu)建了霍亂弧菌膽鹽外排相關(guān)基因的篩選方法,并驗證了該篩選方法是可行的。同時構(gòu)建了霍亂弧菌與有氧生長相關(guān)的細(xì)胞色素c氧化酶基因vca0038和群體感應(yīng)信號分子C-signal合成基因vc1815的框內(nèi)敲除突變株。發(fā)現(xiàn)vca0038缺失突變株的有氧生長以及細(xì)胞色素c氧化酶氧化能力未受明顯影響。而vc1815缺失突變株的生物膜形成等與群體感應(yīng)相關(guān)的性狀與野生型相比也無明顯差別,推測這兩個基因可能不影響霍亂弧菌有氧生長情況下的生長。
[Abstract]:Cholera is an ancient and widespread disease of intense intestinal infection caused by Vibrio cholerae, a gram-negative bacterium. The pathogenicity of Vibrio cholerae is mainly caused by the coregulation of cholera toxin and toxin. Bile salt is a part of bile that helps digest fat in the body. It is also a scale removing agent that destroys cell membranes and kills cells. Vibrio cholerae can tolerate certain concentrations of bile salt. In this study, transposons were randomly inserted into the genome to screen a pair of crude bile salts. Mutant strains of Vibrio cholerae with significantly decreased tolerance to sodium cholate and sodium deoxycholate, It was found by gene sequencing that this gene was consistent with the bile salt efflux pump reported in the literature, which indicated that the method of screening bile salt efflux related genes by random inactivation of transposon was feasible. This system can be used to screen other possible bile salt efflux related genes. Cytochrome c oxidase is the terminal oxidase of electron transfer chain. It gets electrons from cytochrome c and reduces oxygen to water. Copper ion presenting gene scol encodes Sc01 protein in yeast. In Bacillus subtilis and other microorganisms, copper ions are transferred to the copper ion redox center of cytochrome c oxidase. In Vibrio cholerae, vca0038 is assumed to be the scol gene. After obtaining the deletion mutants of the gene, The growth rate of Vibrio cholerae mutant in LB liquid medium and M9 liquid medium did not decrease, and the oxidation ability of cytochrome c oxidase and tolerance to hydrogen peroxide did not decrease. It is speculated that this gene has no effect on the oxidation ability of cytochrome c oxidase of Vibrio cholerae. Vibrio cholerae has three sets of colony sensing systems: 1 / Cqs / A / S system and LuxS / PQ system and VarS/VarA-CsrA/BCD system. They regulate virulence gene expression of Vibrio cholerae. The group sensing signal molecule C-Signal accumulates during aerobic growth of myxobacteria Myxococcus xanthus. This suggests that in Vibrio cholerae, The product of the hypothetical C-signal synthetic gene vc1815 may have an effect on the colony sensing system of Vibrio cholerae. The biofilm forming ability of Vibrio cholerae mutant strain was not significantly affected by the deletion of the gene in this study. Its virulence co-regulated the synthesis ability of TCP did not decrease, nor did the colonization ability in the intestinal tract of newborn mice. It is speculated that this gene has no effect on the colony sensing system of Vibrio cholerae (Vibrio cholerae). In this study, we constructed a screening method for bile salt efflux related genes of Vibrio cholerae by transposon insertion inactivation. At the same time, the cytochrome c oxidase gene vca0038 related to aerobic growth of Vibrio cholerae and the gene vc1815 of the colony sensing signal molecule C-signal synthesizer vc1815 were constructed. It was found that the mutant was deficient in vca0038. The aerobic growth and the oxidation ability of cytochrome c oxidase were not significantly affected, but the biofilm formation of vc1815 deletion mutant was not significantly different from that of wild type. It is speculated that these two genes may not affect the growth of Vibrio cholerae during aerobic growth.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378

【共引文獻(xiàn)】

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本文編號：1529618