摘要
目的利用λ-red同源重组系统构建食源性肠炎沙门氏菌(Salmonella enteritidis,SE)CICC21482rpoS基因缺陷株SE/⊿rpoS,观察其在胁迫环境中的生长特性,以探讨rpoS基因的生物学作用。方法通过PCR扩增两侧为肠炎沙门氏菌rpoS基因同源臂,中间为卡那霉素抗性基因(kmr)的打靶片段,再电击转入含质粒pkD46的肠炎沙门氏菌中,替换rpoS基因,筛选阳性转化子SE/⊿rpoS并用PCR法验证。结果成功构建了肠炎沙门氏菌rpoS基因缺陷株。在常规LB培养基中37℃培养的缺陷株和野生株生长曲线差异无统计学意义(P>0.05);SE/⊿rpoS菌株在45℃、2%NaCl和pH4.0应激条件下的生存能力较野生株弱(P<0.05)。结论成功构建SE/⊿rpoS基因缺陷株。该基因在肠炎沙门氏菌克服高温、高渗、酸应激时被诱导表达从而发挥作用。此结果为进一步研究rpoS基因在应激过程中的作用机制提供了基础资料。
Objectives To construct a mutant by knocking out the rpoS gene of Salmonella enteritidis with the help of a λ-red recombinant system,to investigate the growth characteristics of the rpoS knockout mutation in S.enteritidis under stress conditions,and to discuss the biological effects of the rpoS gene.Methods A kanamycin-resistant gene flanked by homologues of the rpoS gene was amplified with PCR and electroporated into S.enteritidis carrying the plasmid pkD46.After the rpoS gene was replaced with the kanamycin-resistant gene,S.enteritidis/rpoS strains were screened and then amplified with PCR.Results An rpoS knockout mutant of S.enteritidis was successfully constructed.There were no significant differences（P0.05）in the growth curves of the rpoS mutant and wild-type strains.S.enteritidis/ rpoS strains had significantly less ability to survive（P0.05）compared to wild-type strains under stress conditions of 45 ℃,2% NaCl,and pH 4.0.Conclusion An rpoS knockout mutant of S.enteritidis was successfully constructed.Expression of the rpoS gene was induced.The rpoS gene plays an important role in the ability of S.enteritidis to overcome stress in the form of high temperatures,high osmolarity,and an acidic environment.This study provided basic data for further study of the mechanism of action of the rpoS gene in response to stress.
出处
《中国病原生物学杂志》
CSCD
北大核心
2013年第8期690-694,共5页
Journal of Pathogen Biology
基金
国家"863"计划项目(No.2011AA100801)
国家自然科学基金项目(No.31071556
30871739)
