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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。
Objective To construct the SE / ⊿rpoS gene of CICC21482rpoS gene of Salmonella enteritidis (SE) by using λ-red homologous recombination system and observe its growth characteristics in stress environment to explore the biological role of rpoS gene. Methods The homologous arms of rpoS gene of Salmonella enteritidis on both sides were amplified by PCR. The targeted fragment of kr-resistant gene (kmr) was amplified in the middle and then electroporated into Salmonella enteritidis harboring pkD46 plasmid to replace rpoS gene. Transformed SE / ⊿rpoS and verified by PCR. Results We successfully constructed the rpoS gene-deficient strain of Salmonella enteritidis. There was no significant difference in the growth curve of wild-type strain and wild strain cultured in conventional LB medium at 37 ℃ (P> 0.05). The survival of SE / ⊿rpoS strain under 45 ℃, 2% NaCl and pH4.0 stress Ability than wild-type weak (P <0.05). Conclusion The SE / ⊿rpoS gene-deficient strain was successfully constructed. The gene plays a role in Salmonella enteritidis overcoming the high temperature, hypertonic, acid stress induced expression. This result provides the basic information for further study of the mechanism of action of rpoS gene during stress.