文献类型：期刊文献

中文题名：溶藻弧菌T3SS exsD基因敲除突变株构建及其表型特征

英文题名：Construction and Characterization of Gene exsDKnock-out Mutant of Vibrio alginolyticus Type Ⅲ Secretion System

作者：王俊霖[1];招茵[1];苏茵茵[1];周诗慧[1];曾福源[1];谢妙[1];王娜[2];简纪常[1];庞欢瑛[1]

机构：[1]广东海洋大学水产学院/广东省水产经济动物病原生物学及流行病学重点实验室/广东省教育厅水产经济动物病害控制重点实验室,广东湛江524088;[2]中国检验检疫科学研究院,北京100176

年份：2021

卷号：41

期号：5

起止页码：35

中文期刊名：广东海洋大学学报

外文期刊名：Journal of Guangdong Ocean University

收录：CSTPCD、、北大核心、北大核心2020

基金：国家自然科学基金(32073015);广东省自然科学基金(2021A1515011078);广东省科技创新战略专项资金(大学生科技创新培育)(pdjh2021b0239);广东海洋大学大学生创新创业训练计划项目(CXXL2020131);广东海洋大学本科生创新团队(CCTD201802)。

语种：中文

中文关键词：溶藻弧菌;Ⅲ型分泌系统;exsD基因;基因敲除;缺失株;表型特征

外文关键词：Vibrio alginolyticus;Type Ⅲ Secretion System (T3SS);exsD gene;gene knockout;deletion strain;characterization

中文摘要：【目的】构建溶藻弧菌(Vibrio alginolyticus)Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)中的调控蛋白操纵子ExsD基因缺失株,研究exsD缺失株表型特征。【方法】采用overlapPCR技术构建缺失株ΔexsD,PCR检测缺失株ΔexsD的遗传稳定性,比较缺失株与野生株之间的生长速度、泳动能力、胞外酶活性、药物敏感性、毒力,分别采用结晶紫和共聚焦电镜方法测定生物膜的差异变化,通过qRT-PCR方法分析exsD的缺失对T3SS效应蛋白Hop转录的影响。【结果】缺失株ΔexsD构建成功;与野生株相比,ΔexsD遗传稳定性和生长速度无显著差别,但泳动能力极显著上升(P <0.01),胞外蛋白酶活性显著上升(P <0.05),形成生物膜能力在24 h时提高(P <0.05);相比野生株,ΔexsD对米诺环素、庆大霉素、卡那霉素、多西环素、新霉素的敏感性从耐药变成中度敏感;ΔexsD缺失株的毒力显著上升,野生株对斑马鱼的半数致死剂量是缺失株的3.68倍。实时荧光定量结果显示,与HY9901野生型相比,exsD基因的缺失增加了效应蛋白Hop的转录表达。【结论】exsD基因对T3SS的致病性起负调控作用,为进一步阐明exsD基因在溶藻弧菌中的作用机理提供一定的理论基础。

外文摘要：【Objective】 To construct the regulatory protein exsD deletion strain of Vibrio alginolyticus Type Ⅲ secretion system, and study the phenotypic characteristics of exsD-deletion strain. 【Methods】Overlap PCR was used to construct the deletion strain ΔexsD, and PCR was used to detect the genetic stability of the deletion mutant strains Δexs D, and the difference of growth rate, swarming motility, extracellular enzymes, drug sensitivity and virulence, crystal violet between mutant strains and wild strain were evaluated, and the change of biofilm formation was determined by confocal microscope, and the transcriptional level of T3SS effector protein Hop was analyzed by qRT-PCR. 【Results】The deletion strain Δexs D was constructed successfully, and the exs D-deletion did not significantly affect the genetic stability and growth rate of V. alginolyticus, but the extracellular protease activity of the deleted strain Δexs D was significantly improved than wild strain(P < 0.05). The ability to form biofilms of the deleted strain Δexs D was enhanced than the wild strain at 24 h(P < 0.05). The exs D-deletion resulted in a markedly increase in swarming motility(P < 0.01);Compared with the wild strain, the sensitivity of the Δexs D strain to minocycline, gentamicin, kanamycin, doxycycline and neomycin changed from resistance to moderate sensitivity. The LD50 result showed that the virulence of the Δexs D-deficient strain is increased, which was 3.68 times compared with the wild strain. Real time fluorescence quantitative showed that compared with the wild strain, the Δexs D strain increased the expression of effector protein Hop.【Conclusion】The exs D gene has a negative regulatory effect on the pathogenicity of T3SS, which provides a theoretical basis for further clarify the mechanism of exs D gene in V. alginolyticus.