溶藻弧菌dldh基因突变株的构建及其生物学功能研究

生物技术通报 ›› 2014, Vol. 0 ›› Issue (10): 161-167.

溶藻弧菌dldh基因突变株的构建及其生物学功能研究

庞欢瑛^{1, 2, 3}, 陈立明^{1, 2, 3}, 黄郁葱^{1, 2, 3}, 简纪常^{1, 2, 3}, 鲁义善^{1, 2, 3}, 吴灶和^{2, 3, 4}

1. 广东海洋大学水产学院,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,湛江 524088; 3.广东省教育厅水产经济动物病害控制重点实验室,湛江 524088; 4. 仲恺农业工程学院,广州 510225

收稿日期:2014-05-30 出版日期:2014-10-20 发布日期:2014-10-17
作者简介:庞欢瑛,女,博士,讲师,研究方向：水产经济动物病害
基金资助:
国家科技支撑计划（2012BAD17B02,2012 BAD17B03）,广东省自然科学基金项目（S2013040014562）

Construction and Characterization of a Mutant of Vibrio alginolyticus ZJ03 Δdldh Strain

Pang Huanying^{1, 2, 3}, Chen Liming^{1, 2, 3}, Huang Yucong^{1, 2, 3}, Jian Jichang^{1, 2, 3}, Lu Yishan^{1, 2, 3}, Wu Zaohe^{2, 3, 4}

1. Fisheries College of Guangdong Ocean University,Zhanjiang 524088; 2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,Zhanjiang 524088; 3. Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions,Zhanjiang 524088; 4. Zhongkai University of Agriculture and Engineering,Guangzhou 510225

Received:2014-05-30 Published:2014-10-20 Online:2014-10-17

摘要/Abstract

摘要： 为揭示二氢硫辛酰胺脱氢酶（Dihydrolipoamide dehydrogenase,DLDH）在溶藻弧菌（Vibrio alginolyticus）致病中的作用,以pRE112自杀质粒为载体,应用插入失活突变法构建dldh基因插入突变株,比较分析了野生株ZJ03与缺失株ZJ03Δdldh在生长、泳动能力、酶活性、生物膜形成、致病性的表现。结果表明,dldh基因的突变,使溶藻弧菌生长的迟缓期延长、泳动能力显著减弱（P<0.05）,酶活性显著降低（P <0.05）,生物膜生成显著减少（P <0.05）,对石斑鱼（Epinephelus coioides）的致病性也明显减弱（P <0.05）。本研究为从细菌能量代谢角度研究弧菌的致病机制提供理论依据。

关键词: 溶藻弧菌, dldh, 缺失株, 生物特性

Abstract: Vibrio alginolyticus, a gram-negative bacterium has been described to be among the most common and economically important aquatic pathogen of fish and shellfish. Dihydrolipoamide dehydrogenase （DLDH）is homodimeric flavoproteins that catalyse the NAD⁺ dependent reoxidation of dihydrolipoamide in a number of multienzyme complexes. This enzyme classically involved in energy metabolism. To identify DLDH’s role during infection, dldh gene deletion strain （ZJ03Δdldh）were constructed using the insertion mutation method. With the pRE112 suicide plasmid as the carrier, the Overlap PCR and homologous recombination technology were used to construct the mutant strains. Furthermore, the changes of the physiology and pathogenicity of ZJ03Δdldh mutant strains compared with the strain ZJ03, such as growth, swarming ability, biofilm and LD50 were investigated. The growth curve showed that the mutant grew slowly in lag phase compared to the wild-type strain, and the swarming ability, enzyme activity and biofilm formation of ZJ03Δdldh mutant strains showed significant reduced（P<0.05）. The fish lethal test showed that the virulence of the ZJ03Δdldh mutant strains was 10² times lower than the ZJ03 strain. In conclusion, the ZJ03Δdldh mutant strains were constructed successfully, and this study showed that enzymes classically involved in energy metabolism may play an important role in pathogenic mechanism of V. alginolyticus.

Key words: Vibrio alginolyticus, dldh, mutant, Biological characteristics

庞欢瑛, 陈立明, 黄郁葱, 简纪常, 鲁义善, 吴灶和. 溶藻弧菌dldh基因突变株的构建及其生物学功能研究[J]. 生物技术通报, 2014, 0(10): 161-167.

Pang Huanying, Chen Liming, Huang Yucong, Jian Jichang, Lu Yishan, Wu Zaohe. Construction and Characterization of a Mutant of Vibrio alginolyticus ZJ03 Δdldh Strain[J]. Biotechnology Bulletin, 2014, 0(10): 161-167.

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