文章摘要
利用mini-Tn10转座子文库筛选鳗弧菌M3发生表型变化的基因
Construction of a mini-Tn10 transposon library to identify genes associated with several phenotypes of Vibrio aguillarum M3
投稿时间:2019-04-26  修订日期:2019-07-02
DOI:
中文关键词: 鳗弧菌  mini-Tn10转座子  突变文库  表型  基因
英文关键词: VibrioSanguillarum  mini-Tn10 transposon  mutant library  phenotype
基金项目:
作者单位E-mail
李倩 上海海洋大学 水产与生命学院 840373607@qq.com 
李贵阳 黄海水产研究所  
李杰 黄海水产研究所  
莫照兰 上海海洋大学 水产与生命学院 上海 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 山东 青岛 mozl@ysfri.ac.cn 
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中文摘要:
      摘要 鳗弧菌(Vibrio anguillarum)的运动能力、菌膜形成能力、胞外蛋白酶产生能力等表型特征的变化与细菌的致病性相关。为了寻找影响鳗弧菌表型变化的基因,本研究使用转座子mini-Tn10(pLOF/Kana)构建了鳗弧菌M3突变株文库,筛选影响表型变化的菌株及相关基因,证明了这些表型变化的突变子与毒力存在一定的相关性。对M3突变文库的1152突变子进行筛选,获得泳动能力改变的突变子1个(编号为6G_1),酪蛋白酶活性发生改变的突变子3个(编号为5A_11,7B_12,7E_12),明胶酶活性发生改变的突变子1个(编号为7H_1),以及菌膜形成能力发生明显变化的突变子3个(编号为5E_2、6A_2、6E_12)。对转座子插入位点进一步分析显示,一个磷酸二酯酶相关基因的突变引起泳动能力增强(P<0.05),leuD、rseB和thiQ的突变引起酪蛋白酶活性明显减弱(P<0.05),potD的突变引起明胶蛋白酶活性显著减弱(P<0.05),leuO、ilvH和grpB的突变引起菌膜形成能力明显减弱(P<0.05)。对这些表型变化的突变子进行毒力感染,发现野生型M3是编号6G_1的半数致死剂量(LD50)的2.04倍,该突变子毒力相对增强。编号为5A_11,7B_12,7E_12 的突变子LD50分别为野生型M3的2.96倍,3.25倍,3.36倍。编号7H_1的LD50是野生型M3的1.25倍,编号5E_2、6A_2和6E_12 的LD50分别为野生型M3的3.34倍,4.08倍,1.84倍,这些突变子毒力相对减弱。这些结果为进一步研究鳗弧菌的发病机制奠定了基础。
英文摘要:
      Abstract: The phenotypic characteristics of Vibrio anguillarum were related to the pathogenicity of bacteria, such as the swimming motion, the ability of membrane formation and extracellular protease production. In order to identify the genes affecting the phenotypic changes of V. anguillarum, this study used Transposon mini-Tn10 (pLOF/Kana) to construct a library of V. anguillarum M3 mutant strains and to screen the strains and related genes that affect phenotypic changes. It is proved that there is a certain correlation between the mutants of these phenotype changes and the virulence. The mutations of 1152 strains of M3 mutant library were screened, and the mutant strains with significant changes are in swimming ability (strain 6G_1), casein enzyme activity (strains 5A_11, 7B_12, 7E_12), gelatin enzyme activity (strain 7H_1) and biofilm formation ability (strains 5E_2, 6A_2, 6E_12). Further analysis showed that mutations in a phosphodiesterase related gene causes increased swimming capacity(P<0.05), mutations in leuD, rseB and thiQ lead to a significant weakening of protease activity (P<0.05), the mutation of potD results in a significant decrease in protease activity (p<0.05), meanwhile, mutations in leuO, ilvH and grpB lead to a significant decrease in the formation capacity of the bacteria (P<0.05). We have a virulent infection of these mutant strains, found that LD50 of wild type M3was 2.04 times higher than that of the number 6G_1, and that the virulence was relatively increased. Numbered 5A_1,7B_12,7E_12 LD50 are 2.96 times, 3.25 times, and 3.36 times higher than wild-type M3, respectively. The LD50 with the number 7H_1 is 1.25 times that of wild M3, and the LD50 with the numbers 5E_2、6A_2 and 6E_12 are 3.34 times, 4.08 times and 1.84 times than wild M3, respectively. These results lay a foundation for further study on the pathogenic mechanism of V.anguillarum.
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