摘要
为构建调节蛋白H-NS及双组分信号转导系统CpxAR的单、双基因缺失株,本研究以大肠杆菌ATCC25922为研究对象,以pKD4质粒为模板,分别扩增出含kan^r基因的线性打靶片段,在pKD46质粒的辅助及L-阿拉伯糖的诱导下进行Red同源重组,使线性打靶片段替换大肠杆菌ATCC25922中的cpxR、hns基因,再用pCP20质粒消除FRT位点间的kan^r基因,构建单基因缺失株△cpxR和△hns。接着以△cpxR为研究对象,用同样方法敲除hns基因,构建双基因缺失株△cpxR△hns。最后将重组表达质粒cpxR-pBAD/HisA、hns-pBAD/HisA电转入上述3种缺失株中,制备回补菌株△cpxR/pcpxR、△hns/phns、△cpxR△hns/pcpxR和△cpxR△hns/phns。结果表明,利用该重组系统成功敲除了大肠杆菌ATCC25922中cpxR、hns基因,构建了单、双基因缺失株△cpxR、△hns和△cpxR△hns以及它们的回补菌株,为研究H-NS及CpxAR互作调控IncFⅡ质粒接合作用的分子机制提供了有力的工具。
The aim of this study was to construct single and double gene deletion strains of H-NS regulatory protein and CpxAR two-component signal transduction system.The cpxR and hns gene were knocked out from ATCC25922 strain via λ-Red homologous recombinant system with the DNA fragment amplified from the plasmid pKD4 by PCR,which contained kanamycin resistance gene(kan^r) and homologues arms of the cpxR and hns genes.Then,the double gene deletion strain(△cpxR△hns) was constructed.In addition,the complemental strains△cpxR/pcpxR,△hns/phns,△cpxR△hns/pcpxR,and △cpxR△hns/phns were prepared through introducting the expressing plasmid cpxR-pBAD/HisA,hns-pBAD/HisA into the single/double deletion strains.The results showed that △cpxR,△hns,△cpxR△hns,△cpxR/pcpxR,△hns/phns,△cpxR△hns/pcpxR,and △cpxR△hns/phns were successfully constructed,which provides powerful tools for studying the molecular mechanism of H-NS and CpxAR interaction in regulating IncFⅡ plasmid conjugation.
作者
胡慧慧
孙亚伟
李文娅
邝启红
孙华润
吴华
胡功政
苑丽
HU Hui-hui;SUN Ya-wei;LI Wen-ya;KUANG Qi-hong;SUN Hua-run;WU Hua;HU Gong-zheng;YUAN Li(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;College of Animal Science and Technology,Henan Institute of Science and Technology,Xinxiang,Henan 453001,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2020年第1期116-121,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31772800)
作者简介
胡慧慧(1993-),女,硕士;通讯作者:苑丽,E-mail:yuanli-hn@163.com。
