利用mini-Tn10转座子文库筛选鳗弧菌M3表型发生变化的基因

为了寻找影响鳗弧菌(Vibrio anguillarum)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和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显著变化的突变子3个(编号为5E_2、6A_2和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突变引起泳动能力增强(P<0.05)，leuD、rseB和thiQ突变引起酪蛋白酶活性显著减弱(P<0.05)，potD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6G_1突变子的半数致死剂量(Lethal dose 50%，LD50)的2.04倍，该突变子毒力相对增强。5A_11、7B_12和7E_12的突变子LD50分别为野生型M3的2.96、3.25和3.36倍。7H_1的LD50是野生型M3的1.25倍，5E_2、6A_2和6E_12的LD50分别为野生型M3的3.34、4.08和1.84倍，这些突变子毒力相对减弱。本研究结果为进一步阐明鳗弧菌的发病机制提供了理论基础。

Construction of A mini-Tn10 Transposon Library to Identify Genes Associated with Several Phenotypes of Vibrio aguillarum M3

The phenotypic characteristics of Vibrio anguillarum are related to the pathogenicity of the bacteria, such as swimming motion, ability of membrane formation, and extracellular protease production. To identify the genes affecting phenotypic changes in 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 mutants causing these phenotypic changes and the virulence. Mutations of 1152 strains of M3 mutant library were screened, and mutant strains with significant changes in swimming ability (strain 6G_1), casein enzyme activity (strains 5A_11, 7B_12, and 7E_12), gelatin enzyme activity (strain 7H_1), and biofilm formation ability (strains 5E_2, 6A_2, and 6E_12) were noted. Further analysis revealed that a phosphodiesterase-related gene mutation caused increased swimming capacity (P<0.05), leuD, rseB, and thiQ mutations caused a significant decrease in caseinase activity (P<0.05), and potD mutations caused a significant decrease in gelatinase activity (P<0.05). Moreover, mutations in leuO, ilvH and grpB resulted in a significant decrease in the ability to form bacterial membranes (P<0.05). Moreover, we observed a virulent infection in these mutant strains, which showed that LD50 of wild type M3 was 2.04 times higher than that of 6G_1 and the virulence was relatively increased. Additionally, 5A_11, 7B_12, and 7E_12 LD50 were 2.96 times, 3.25 times, and 3.36 times higher than that of wild-type M3, respectively. The LD50 with the strain 7H_1 was 1.25 times higher than that of wild M3, and the LD50 with the strains 5E_2, 6A_2, and 6E_12 were 3.34, 4.08, and 1.84 times higher than that of wild M3, respectively. These results lay a foundation for further study on the pathogenic mechanism of V. anguillarum.