中国水产科学2017，Vol.24Issue(5)：977-987,11.DOI:10.3724/SP.J.1118.2017.17131
尼罗罗非鱼无乳链球菌基因缺失株△cpsE和△neuA的构建及其生物学特性
Construction and characterization of cpsE-and neuA-deleted mutants of Streptococcus agalactiae isolated from Nile tilapia
摘要
Abstract
To investigate the functions of the capsular polysaccharide synthetic genes cpsE and neuA of Streptococcus agalactiae (GBS) isolated from Nile tilapia (Oreochromis niloticus),two single-gene knockout mutant strains,namely △cpsE and △neuA,were constructed by homologous recombination.Genomic DNA of GBS was used as a template to amplify the up and down homologous fragments of cpsE and neuA,whereas the pSET1 plasmid was used as a template to amplify the chromosomal chloramphenicol resistance gene (cat).Two recombinant gene knockout plasmids,pSET4s-cpsE and pSET4s-neuA,both containing cat,were constructed by the In-Fusion(R) polymerase chain reaction (PCR) method.The recombinant plasmids pSET4s-cpsE and pSET4s-neuA were transformed into wild-type GBS by electroporation.Double-crossover and plasmid loss strains were obtained by changing the culture temperature.Finally,△cpsE and △neuA were screened for chloramphenicol resistance and the mutations were confirmed by PCR,real-time PCR,and DNA sequencing.To characterize △cpsE and △neuA,their growth rate,capsule thickness,capsular sialic acid content,and virulence were compared with those of wild-type GBS.The results showed that the growth rates of the wild-type,△cpsE,and △neuA strains did not significantly differ.However,the capsule thickness,capsular sialic acid content,and virulence of △cpsE and △neuA were significantly lower than those of the wild-type strain.Further research suggested that cpsE is the critical synthetic gene of the capsular polysaccharide of GBS,whereas neuA is important for capsular polysaccharide sialylation.The deletion of cpsE and neuA not only significantly reduced the capsular sialic acid content of GBS isolated from fish,but also significantly impaired its virulence.关键词
尼罗罗非鱼/无乳链球菌/荚膜多糖合成基因/基因敲除/生物学特性Key words
Oreochromis niloticus/Streptococcus agalactiae (GBS)/capsular polysaccharide synthetic gene/knockout mutants/characterization分类
农业科技引用本文复制引用
师红亚,董浚键,张德锋,孙成飞,田园园,曾庆凯,卢迈新,叶星..尼罗罗非鱼无乳链球菌基因缺失株△cpsE和△neuA的构建及其生物学特性[J].中国水产科学,2017,24(5):977-987,11.基金项目
国家自然科学基金项目(NSFC)(31272688) （NSFC）
现代农业产业技术体系建设专项资金项目(CARS-48) （CARS-48）
中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金项目(2017YH-ZC06). （2017YH-ZC06）
