溶藻弧菌kdpD基因敲除突变株的构建及其表型特征  

Construction and characterization of the kdpD gene knock-out mutant of Vibrio alginolyticus

作　　者：张燕飞[1,2,3] 庞欢瑛[1,2,3] 简纪常[1,2,3] 鲁义善[1,2,3] 吴灶和[2,3,4] 

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524088 [3]广东省水产经济动物病害控制重点实验室,广东湛江524088 [4]仲恺农业工程学院,广东广州510225

出　　处：《微生物学通报》2015年第9期1770-1778,共9页Microbiology China

基　　金：广东省海洋经济创新发展区域示范专项项目(No.GD2012-B01-004);国家科技支撑项目(No.2012BAD17B03);广东高校国际合作创新平台项目(No.2013gjhz0008)

摘　　要：【目的】探究kdpD基因对溶藻弧菌生物学特性的影响。【方法】采用Overlap PCR和同源重组技术,结合正负向筛选,构建kdpD基因无标记基因框内敲除突变株,比较kdpD突变株和野生株HY9901在生长速率、胞外蛋白酶活性以及毒力等方面的差异。【结果】成功构建溶藻弧菌kdpD基因敲除突变株。体外实验表明,kdpD的缺失对溶藻弧菌的生长曲线和胞外蛋白酶活性的影响不明显,但是突变株的泳动能力和生物被膜形成能力出现下降。斑马鱼致病性试验结果显示,突变株毒力下降了8.84倍。【结论】kdpD基因参与调控溶藻弧菌的泳动能力、被膜形成和毒力,但不影响生长速率和胞外蛋白酶活性。[Objective] To investigate the role of kdpD gene in Vibrio alginolyticus, a kdpD gene deletion mutant was generated. [Methods] Combining with chloramphenicol （Cm） and sucrose screening, we constructed an in-flame deletion mutant of kdpD using overlap PCR and homologous recombination technology. We then investigated the effect of the mutant on its growth, ECPase activity and virulence in fish. [Results] The mutant △kdpD was successfully constructed. The mutant △kdpD showed no differences of ECPase activity and growth from the wild-type strain. But a decrease in swarming ability and biofilm formation was found. Moreover, virulence assays with zebrafish model confirmed that virulence of the mutant declined by 8.84 times. [Conclusion] The kdpD gene could regulate swarming ability, biofilm formation and virulence, but it has no effect on growth and ECPase activity.

关 键 词：溶藻弧菌 KDP D 基因敲除 表型 

分 类 号：S941.4[农业科学—水产养殖]