Construction and characterization of cpsE -and neuA -deleted mutants of Streptococcus agalactiae isolated from Nile tilapia

Abstract

PDF HTML阅读 XML下载 导出引用 引用提醒 尼罗罗非鱼无乳链球菌基因缺失株I>cpsE和I>neuA的构建及其生物学特性 DOI: 作者: 作者单位: 1. 中国水产科学研究院 珠江水产研究所, 农业部热带亚热带水产资源利用与养殖重点实验室, 广东 广州 510380;2. 上海海洋大学 水产与生命学院, 上海 201306 作者简介: 师红亚(1990-),女,硕士研究生,从事水产生物技术研究.E-mail:839782122@qq.com;董浚键(1987-),男,助理研究员,从事水产生物技术研究.E-mail:dongjj@prfri.ac.cn;shantoumaliugan@gmail.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（NSFC）（31272688）；现代农业产业技术体系建设专项资金项目（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）. Construction and characterization of cpsE-and neuA-deleted mutants of Streptococcus agalactiae isolated from Nile tilapia Author: Affiliation: 1. Key Laboratory of Tropical & Subtropical Fisheries Resource Application & Cultivation, Ministry of Agriculture;Pearl River Fisheries Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China;2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为探究尼罗罗非鱼无乳链球菌（GBS）荚膜多糖合成基因对菌株生物学特性的影响，本研究利用同源重组的方法，构建了GBS的的单基因缺失突变株。具体方法为：用Infusion-PCR的方法分别构建带有氯霉素抗性基因的基因敲除重组质粒pSET4s-。将构建好的质粒电转化入GBS感受态细胞中，通过改变培养温度实现双交换和质粒丢失，最后经氯霉素抗性筛选获得疑似敲除株。通过菌落PCR、RT-PCR及DNA测序等方法对疑似敲除株进行验证。结果显示GBS的两个突变株I>cpsE和I>neuA被成功构建。在此基础上，通过生物学功能分析比较基因缺失突变株I>cpsE、I>neuA与野生株在菌株生长速率、荚膜多糖厚度、唾液酸含量和毒力方面的差异。结果发现缺失突变株I>cpsE和I>neuA的生长速度与野生株无显著差异，但荚膜多糖厚度、唾液酸含量和菌株毒力均显著低于野生株。进一步研究显示，是鱼源GBS荚膜多糖合成的关键基因，基因则是荚膜多糖唾液酸化的关键基因，它们的缺失导致了GBS荚膜唾液酸含量的降低，且显著降低了菌株的毒力。 Abstract:To investigate the functions of the capsular polysaccharide synthetic genes (GBS) isolated from Nile tilapia (cpsE and , were constructed by homologous recombination. Genomic DNA of GBS was used as a template to amplify the up and down homologous fragments of , whereas the pSET1 plasmid was used as a template to amplify the chromosomal chloramphenicol resistance gene ( and pSET4s-?/SUP> polymerase chain reaction (PCR) method. The recombinant plasmids pSET4s- were transformed into wild-type GBS by electroporation. Double-crossover and plasmid loss strains were obtained by changing the culture temperature. Finally, were screened for chloramphenicol resistance and the mutations were confirmed by PCR, real-time PCR, and DNA sequencing. To characterize , 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, strains did not significantly differ. However, the capsule thickness, capsular sialic acid content, and virulence of were significantly lower than those of the wild-type strain. Further research suggested that is the critical synthetic gene of the capsular polysaccharide of GBS, whereas is important for capsular polysaccharide sialylation. The deletion of not only significantly reduced the capsular sialic acid content of GBS isolated from fish, but also significantly impaired its virulence. 参考文献 相似文献 引证文献