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目的对铜绿假单胞菌(Pseudomonas aeruginosa,PA)野生型PAO1中与环鸟苷二磷酸(C-di-GMP)代谢相关的4个具有GGDEF-EAL结构域基因(PA0285、PA0575、PA5295、PA5442)进行初步研究。方法分析4个基因的转座子突变菌株进行生物膜(Biofilm)及泳动性(Motility);利用PCR对4个基因的催化结构域进行克隆并构建原核表达载体,经IPTG诱导后检测重组蛋白的表达情况。结果生物膜分析显示,PA0285、PA0575、PA5442的转座子突变体较野生型PAO1间生物膜合成发生变化,其A值差异有统计学意义(P=0.000,P<0.05);PA5295转座子突变体与PAO1间生物膜合成差异无统计学意义(P=0.232,P>0.05);泳动性分析显示,PA0285、PA0575、PA5442的转座子突变体菌落直径与PAO1比较差异有统计学意义(P=0.000,P<0.05),PA5295的转座子突变体与PAO1间比较差异无统计学意义(P=0.083,P>0.05)。4个基因的催化结构域原核表达载体构建成功,并在大肠埃希菌中表达目的蛋白。结论初步分析表明PA0285、PA0575、PA5442均影响细菌的生物膜合成及运动性,为4基因的功能研究奠定了基础。
OBJECTIVE: To investigate the effect of GGDEF-EAL domain gene (PA0285, PA0575, PA5295, PA5442) on the metabolism of cyclic guanosine diphosphate (C-di-GMP) in wild-type PAO1 of Pseudomonas aeruginosa ) Conducted a preliminary study. Methods Four transposon mutant strains were analyzed for biofilm and motility. The catalytic domain of four genes was cloned by PCR and the prokaryotic expression vector was constructed. After induced by IPTG, the recombinant protein The expression of the situation. Results Biofilm analysis showed that the transposon mutants of PA0285, PA0575 and PA5442 changed more than the wild-type PAO1 biofilm, and the difference of A value was statistically significant (P = 0.000, P <0.05); PA5295 transposon There was no significant difference in biofilm synthesis between the mutant and PAO1 (P = 0.232, P> 0.05). The analysis of motility showed that there was significant difference between the colonies diameter of PA0285, PA0575 and PA5442 transposon mutant and PAO1 (P = 0.000, P <0.05). There was no significant difference between PA5295 transposon mutants and PAO1 (P = 0.083, P> 0.05). The prokaryotic expression vector of the 4 genes was successfully constructed and the target protein was expressed in Escherichia coli. Conclusion Preliminary analysis shows that PA0285, PA0575 and PA5442 both affect the biofilm synthesis and motility of bacteria, which lays the foundation for the functional study of 4 genes.