Preliminary Study On The Construction And Function Of Aeromonas Vaginalis TH0426 RpoN And RpoE Gene Deletion Strain

Aeromonas veroni(A.veronii)is an important pathogen of human-beast-aquatic zoonosis.Studies have shown that the recent virulence has been increasing year by year and has been paid more and more attention by scholars.However,there are few studies on the virulence of A.veronii,and the study of virulence genes can help to provide a theoretical basis for the study of the pathogenic mechanism of A.veronii.The research on rpoN gene was first discovered in 1985.In recent years,research reports on rpoN have been increasing year by year.It can control the transcriptional expression of multiple functional genes,including nitrogen metabolism,counter-reaction,carbon source utilization,exercise and virulence.Expression plays a vital role in many bacteria.Therefore,the rpoN gene was selected as the knockout target,and the deletion strain ?rpoE of A.veronii TH0426 strain rpoN and its complementary strain C-rpoE were constructed by suicide plasmid-based homologous recombination technique,and the above bacteria were partially biologically studied.Characteristic analysis showed that there was no obvious difference in growth curve and hemolytic activity.In the morphology of the colony,the ?rpoN of the deletion strain showed obvious wavy edge,and the complementary strain C-rpoN recovered to the surface of the wild plant with smooth surface and neat edges.The LD50 of zebrafish showed that the LD50 of the ?rpoN was 22.9 times higher than that of the wild type,the LD50 of the mouse increased by 3.06 times,and the virulence decreased significantly.The biofilm formation ability showed that the ?rpoN was deleted.Compared with the wild strain,the difference was 11 times,the replenished strain was enhanced,but it was not restored to the wild strain level;the adhesion and invasion of the cells and the toxicity to EPC were also decreased;the swimming ability test showed that the wild strain TH0426 has flagella and has swimming ability,while the swimming ability of the missing strain ?rpoN is basically lost.It can be observed by further flagellar staining test results.TH0426 wild strain having a polar flagellum,each cell has one,but no significant deletion mutant ? rpoN flagellin,flagellin complementary strain fracture small amount C-rpoN view.rpoE is a key factor in the defense against adverse reactions in the unfavorable environment.It regulates the activity of the gene to regulate the activity of the bacteria,assists the bacteria to produce a thermal response,and resists the harsh environment,which is vital for the survival of the bacteria.Impact.Therefore,rpoE was knocked out by the same method.The results showed that the hemolytic activity of the ?rpoE was unchanged compared with the wild type,and the colony morphology did not change significantly,but the growth rate was slower than that of the wild strain;the biofilm was missing.The ?rpoE strain was 3.7 times lower than that of the wild type strain;the adhesion and invasion ability was also lower than that of the wild type strain.The LD50 of the deletion strain ?rpoE was 82.3 times higher than that of the wild type strain,and the LD50 of the mouse was increased by 5.22 times;Toxicity test results showed that the ?rpoE of the deletion strain was significantly less toxic to EPC than the wild strain TH0426;the exercise ability test showed that the ?rpoE lacked the motility in the semi-solid medium,and the ?rpoE was absent in the flagellar staining results.Significant flagella.In summary,this study constructed the A.veronii TH0426 strain rpoN and rpoE deletion strains and complementary strains and compared their biological differences with wild strains,and speculated the function of rpoN and rpoE.The research on the pathogenesis of veronii laid the foundation.