CAJ | 학술논문

目的通过表型实验分析Hfq蛋白对霍乱弧菌环境适应能力的影响,并通过分子实验验证Hfq对环境生存及致病相关基因转录的调控.方法基于自杀质粒缺失霍乱弧菌的hfg基因,并构建相应回补株；通过表型实验,比较霍乱弧菌野生株和hfg突变株在细菌运动能力、生物膜形成、外膜抗性和氧化应激能力上的差异；应用逆转录荧光定量PCR(RT-qPCR)技术对Hfq可能调控的相关基因的转录水平进行比较.结果成功构建霍乱弧菌hfg缺失株及回补株,表型实验表明hfq突变株的菌株游动性降低,生物膜形成能力增强,对十二烷基磺酸钠(SDS)和庆大霉素的耐受性显著下降,并且与野生株相比,hfg突变株对H2O2刺激更为敏感.RT-qPCR实验证明Hfq对霍乱弧菌鞭毛合成、胞外多糖合成、外膜蛋白以及氧化应激相关基因的转录有调控作用.结论 Hfq蛋白作为RNA伴侣分子,能够通过调控一些生存及致病相关基因的转录进而影响霍乱弧菌的生物膜形成、氧化应激反应、抗生素抗性等能力,表明Hfq可能与其他调控子一起,在霍乱弧菌的生存、代谢以及致病因子转录的协调控制上起关键作用.
목적 통과표형실험분석Hfq단백대곽란호균배경괄응능력적영향,병통과분자실험험증Hfq대배경생존급치병상관기인전록적조공.방법 기우자살질립결실곽란호균적hfg기인,병구건상응회보주；통과표형실험,비교곽란호균야생주화hfg돌변주재세균운동능력、생물막형성、외막항성화양화응격능력상적차이；응용역전록형광정량PCR(RT-qPCR)기술대Hfq가능조공적상관기인적전록수평진행비교.결과 성공구건곽란호균hfg결실주급회보주,표형실험표명hfq돌변주적균주유동성강저,생물막형성능력증강,대십이완기광산납(SDS)화경대매소적내수성현저하강,병차여야생주상비,hfg돌변주대H2O2자격경위민감.RT-qPCR실험증명Hfq대곽란호균편모합성、포외다당합성、외막단백이급양화응격상관기인적전록유조공작용.결론 Hfq단백작위RNA반려분자,능구통과조공일사생존급치병상관기인적전록진이영향곽란호균적생물막형성、양화응격반응、항생소항성등능력,표명Hfq가능여기타조공자일기,재곽란호균적생존、대사이급치병인자전록적협조공제상기관건작용.
Objective To characterize Hfq-dependent phenotypes in stress response and to dissect Hfq-dependent transcription of virulence genes and stress-responsive genes in Vibrio cholera.Methods The hfq null mutant strain (△hfq) and the complemented mutant strain (△hfq/pUC18-hfq) were constructed from the wild-type Vibrio cholera.Comparisons on the motility,biofilm formation,growth under various oxygen-supplying conditions,outer membrane resistance,and sensitivity to oxidative stress were analyzed between the wild type strain and the mutant strains.Reverse-transcript fluorescence quantitative PCR (RT-qPCR) was used to determine the transcriptional levels of target genes in the above mentioned strains.Results △hfq and △hfq/pUC18-hfq strains were successfully constructed.The motility,outer membrane resistance and sensitivity to oxidative stress were reduced,but biofilm formation was enhanced in the hfq null mutant strain.RT-qPCR testified that Hfq had regulation effects on gene transcription for forming falagellum,extracellular polysaccharide,outer membrane protein and oxidative stress in Vibrio cholera.Conclusion As a RNA chaperone,Hfq could affect Vibrio cholera in its biofilm formation,resistance to oxidative stress and antibiotics resistance through regulating the transcription of multiple metabolic genes and virulence genes,which indicates that Hfq,combined with other regulators,may play a key role in the complex regulation of metabolic genes and virulence genes.