Studies Of RecA Gene Function Of Riemerella Anatipestifer RA-GD Strain

Riemerella anatipestifer is a flagellum-free,spore-free,Gram-negative bacterium stained with two poles.The infectious disease of domestic ducks caused by this bacterium is called duck infectious serositis,and the extensive cellulous inflammation such as fibrinous pericarditis is a clinicopathological feature.The disease is widely distributed throughout the world,with high morbidity and mortality,and is one of the major infectious diseases that cause significant losses in the duck industry.The bacterial DNA damage-inducing reaction,also known as the SOS response,refers to an important protective mechanism for bacteria to cope with genomic DNA damage.The RecA/LexA protein plays a key regulatory role in the induction of SOS responses in many bacteria.To study the function of the RecA gene and its role in the DNA damage repair response(SOS response),to help understand the weakening of bacterial virulence and evolution,reduce bacterial pathogenicity,and help reduce and control the increasingly severe bacterial resistance,and play a role in the development of new antibacterial small molecules and biological agents,reducing the use of chemical drugs to control pathogens.First,on the basis of homologous recombination,the researchers constructed the deletion strain RA-GD?RecA through the suicide plasmid PRE112-RecA-Erm.On the basis of the deletion strain RA-GD?RecA,the complement plasmid Pcp29-Mcs-Ori-Psr-RecA was constructed by sequentially introducing the multiple cloning site(Mcs),the replication region(Ori),the promoter(Psr)and the RecA gene sequence into the complement plasmid Pcp29.Thus,the complement strain cRA-GD?RecA was constructed by conjugative transfer with the parental strain RA-GD.Secondly,the above three strains of R.anatipestifer(the parental strain RA-GD,the deletion strain RA-GD?RecA and the complement strain cRA-GD?RecA)were respectively subjected to biological characteristics such as growth curve,susceptibility test and ducklings virulence test.The results showed that the RecA gene did not affect the growth rate of R.anatipestifer RA-GD;the RecA gene played a role in reducing the sensitivity of R.anatipestifer to gentamicin sulfate;compared with RA-GD,the deletion of RecA gene significantly reduced the lethality rate of RA-GD?RecA in ducklings,indicating that RecA gene may be involved in the influence and weaken the expression of virulence factors of R.anatipestifer.Finally,the transcriptional level and protein expression level of RecA gene were compared before and after damage between parental strain RA-GD and complement strain cRA-GD ? RecA by UV damage factors.The results showed that within a certain limit,the transcription level of the RecA gene increased with the prolongation of UV irradiation time.UV factor can trigger a DNA damage response(SOS response).The UV damage assay indicated that the RecA gene may play a positive regulatory rolein the DNA damage repair response(SOS response)of R.anatipestifer RA-GD strain.Western blotting indicated that,UV may not be involved in affecting the expression of RecA protein in the parent strain RA-GD and the complement strain cRA-GD?RecA.