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目的构建铜绿假单胞菌(PA)小RNAphrs基因缺失株与回复株,观察phrs在铜绿假单胞菌生长和生物膜形成中的作用。方法应用λ-Red重组系统构建phrs缺失株PA27853Δphrs、回复株PA27853Δphrs-comp和对照株PA27853Δphrs-control,绘制细菌生长曲线,观察phrs对细菌生长的影响;荧光染色法定量分析细菌生物膜形成能力变化。结果成功获得phrs基因缺失株和回复株,RT-PCR结果显示,phrs基因在缺失突变株中不表达,在回复株中重新表达;phrs不影响细菌的生长速度,基因敲除后细菌生物膜形成能力明显下降,而回复株则能形成明显生物膜。结论利用λ-Red重组技术成功构建出PA25783Δphrs,phrs对细菌生物膜的形成具有重要的调控作用。
Objective To construct the deletion and recovery strains of small RNAphrs genes of Pseudomonas aeruginosa (PA), and to observe the role of phrs in the growth and biofilm formation of Pseudomonas aeruginosa. Methods The phrs deletion strain PA27853Δphrs, PA27853Δphrs-comp and the control strain PA27853Δphrs-comp were constructed by λ-Red recombination system. The growth curve of bacteria was drawn and the effects of phrs on the growth of bacteria were observed. The changes of bacterial biofilm formation ability were analyzed by fluorescence staining. Results The phrs gene deletion and recovery strains were successfully obtained. The results of RT-PCR showed that phrs gene was not expressed in the deletion mutant and re-expressed in the recovery strain. Phrs did not affect the growth rate of the bacteria, and the bacterial biofilm formation after gene knockout Ability decreased significantly, while the reply strain can form a significant biofilm. Conclusion PA25783Δphrs was successfully constructed using λ-Red recombination technology. Phrs plays an important regulatory role in the formation of bacterial biofilm.