摘要目的 通过Red同源重组法构建qseC基因缺失的大肠杆菌突变株,并探讨qseC基因对大肠杆菌生物被膜形成的影响.方法 PCR扩增两翼与目的基因上下游同源、含有氯霉素抗性基因片段,电击转化人大肠杆菌MC1000,在Red同源重组酶作用下,用含同源臂的氯霉素抗性片段置换目的基因qseC,并利用FLP位点专一性重组将氯霉素抗性基因删除.结晶紫染色半定量法分析qseC基因缺失株生物被膜形成能力的变化.结果 PCR及DNA测序结果表明,qseC基因已被成功敲除.在LB培养基中,qseC基因缺陷株的生长状况与亲株无明显差异.MC1000与MC1000 △qseC基因缺失株生物被膜形成能力半定量A值的结果分别为1.00±0.15和0.47±0.10.结论 成功构建大肠杆菌qseC基因缺失突变株,且qseC基因对细菌生物被膜的形成具有调控作用.

abstractsObjective To construct a qseC-deleted mutant strain of E.coli by Red recombination and to study the effect of qseC gene on biofilm formation in the mutants.Methods The chloramphenicolresistant gene flanked by homologues of target genes was amplified by PCR and electro-transformed into E.coli MC1000.When induced by L-arabinose,the plasmid pKD46 could express three recombinant proteins of λ-prophage,which led to the replacement of target gene(qseC) with chloramphenicol-resistant gene.Then the chloramphenicol-resistant gene was eliminated by FLP-promoted recombination events.The biofilm formation of wild-type and mutant strain was detected by crystal violet staining.Results The qseC-deleted mutant of E.coli was confirmed by various PCR and DNA sequencing.Gene qseC was completely deleted.There was no significant difference in growth ability between the qseC mutant strain and the wild-type strain MC1000.The biofilm formation of wild-type and mutant strain was quantified by crystal violet staining.The absorbance determined with a plate reader at 570 nm was 1.00±0.15 and 0.47±0.10 respectively.Conclusion The qseC-deleted mutant of E.coli was constructed successfully.And the qseC gene plays an important role in regulation of biofilm formation in E.coli.