霍乱弧菌胆盐外排相关基因筛选方法的建立及有氧生长相关蛋白Scol和C-signal的研究

发布时间：2018-02-24 08:46

本文关键词： 霍乱弧菌胆盐有氧生长细胞色素c氧化酶 C-signal　出处：《南京农业大学》2012年硕士论文　论文类型：学位论文

【摘要】：霍乱是一种古老且流行广泛的烈性肠道感染疾病,它是由革兰氏阴性细菌霍乱弧菌所引起的。大量的研究表明,霍乱弧菌的致病能力主要是由霍乱毒素和毒素共调节菌毛引起的。胆盐是胆汁的一部分,在人体内能够帮助消化脂肪。它也是一种去垢剂,能破坏细胞膜从而杀死细胞。霍乱弧菌能够耐受一定浓度的胆盐,说明它有某些基因能够把胆盐外排出细胞或是降解胆盐。本实验采用转座子随机插入基因组的方法筛选到一株对粗胆盐、胆酸钠和脱氧胆酸钠耐受能力显著下降的霍乱弧菌突变株,经基因测序分析发现此基因与文献报道的霍乱弧菌胆盐外排泵一致。说明这种利用转座子随机插入失活来筛选胆盐外排相关基因的方法的建立是可行的,可以通过此体系筛选其他可能存在的胆盐外排相关基因。细胞色素c氧化酶是电子传递链终端氧化酶,它从细胞色素c得到电子,并把氧气还原成水。铜离子递呈基因scol编码的Sc01蛋白在酵母、枯草芽孢杆菌等微生物中起到了把铜离子传递给细胞色素c氧化酶的铜离子氧化还原中心的作用。在霍乱弧菌中vca0038为假设的scol基因。本实通过框内敲除的方法获得该基因的缺失突变株后,霍乱弧菌突变株在LB液体培养基和M9液体培养基中有氧条件下生长速度并未下降,细胞色素c氧化酶氧化能力和对过氧化氢的耐受能力也没有下降。因此,推测该基因对霍乱弧菌的细胞色素c氧化酶氧化能力没有影响。霍乱弧菌有三套群体感应系统：CqsA/S系统、LuxS/PQ系统和VarS/VarA-CsrA/BCD系统。它们对霍乱弧菌的毒力基因表达等调控作用。群体感应信号分子C-Signal在粘细菌Myxococcus xanthus有氧生长时细胞聚集并形成孢子的过程中起作用。这提示在霍乱弧菌中,假设的C-signal合成基因vc1815的产物对霍乱弧菌的群体感应系统可能有所影响。本实验缺失该基因后,霍乱弧菌突变株的生物膜形成能力并未受明显影响,其毒力共调节菌毛TCP的合成能力也未下降,在乳鼠肠道内的定殖能力也未受到影响,推测该基因对霍乱弧菌群体感应系统可能没有影响。本实验通过转座子插入失活的方式构建了霍乱弧菌胆盐外排相关基因的筛选方法,并验证了该筛选方法是可行的。同时构建了霍乱弧菌与有氧生长相关的细胞色素c氧化酶基因vca0038和群体感应信号分子C-signal合成基因vc1815的框内敲除突变株。发现vca0038缺失突变株的有氧生长以及细胞色素c氧化酶氧化能力未受明显影响。而vc1815缺失突变株的生物膜形成等与群体感应相关的性状与野生型相比也无明显差别,推测这两个基因可能不影响霍乱弧菌有氧生长情况下的生长。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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