pvdQ基因对铜绿假单胞菌生物膜形成的影响

Effects of pvdQ Gene on Pseudomonas aeruginosa Biofilm Formation

目的观察pvdQ(PA2385)基因对铜绿假单胞菌生物膜形成的影响。方法构建pvdQ基因表达质粒pME6032-pvdQ,鉴定后,采用电穿孔法将其转入PAO1野生株中,构建pvdQ高表达株。同时将空质粒pME6032采用电穿孔法转入PAO1野生株中,构建pME6032空质粒株,采用Real-time PCR检测各株pvdQ mRNA的表达。采用结晶紫染色法,观察PAO1野生株、pvdQ高表达株和pvdQ突变株培养24、48和72 h时生物膜的生长情况。结果 Real-time PCR证实成功构建pvdQ高表达株。结晶紫染色的结果显示:培养24h时PAO1野生株、pvdQ高表达株和pvdQ突变株形成的生物膜厚度差异无统计学意义(P>0.05);培养48 h和72h时与PAO1野生株比较,pvdQ高表达株生物膜明显变薄(P<0.05),pvdQ突变株生物膜明显增厚(P<0.05)。结论铜绿假单胞菌PAO1野生株、pvdQ突变株和pvdQ高表达株体外形成生物膜的能力有显著性差异,pvdQ基因可能影响铜绿假单胞菌体外形成生物膜的能力。

Objective To investigate the effects of pvdQ（PA2385）gene on Pseudomonas aeruginosa biofilm formation.Methods The plasmid pME6032 containing pvdQ gene was constructed and identified,then transformed into Pseudomonas aeruginosa PAO1 wild type by using the electroporation,building pvdQ overexpression strain（pME6032-pvdQ）.By using the same method,the PAO1pME6032 strain was constructed.The expression of pvdQ mRNA was deteded by using real-time PCR.Biofilm formation was quantified by using crystal violet（CV）assay,and biofilm development was monitored at different time points（24,48,and 72 h）.Results Real-time PCR confirmed that the pME6032-pvdQ strain was successfully constructed.There was no significant difference in the biofilm formation among PAO1,pME6032-pvdQ strains,and pvdQ mutant strains（PAO1ΔpvdQ）at 24 h（P0.05）,but at 48 and 72 h biofilm was thicker in PAO1ΔpvdQ strains than in PAO1 wild type（P0.05）,and biofilm was thinner in pME6032-pvdQ strains than in PAO1 wild type（P0.05）.Conclusion There was significant difference in biofilm formation in vitro among PAO1,pME6032-pvdQ strains,and pvdQ mutant strains.pvdQ gene may play an important role in the formation of Pseudomonas aeruginosa biofilm.