Construction And Characterization Of Riemerella Anantipestifer Strain ECFs Sigma Gene Deletion

Riemerella anatipestifersis which was caused by Riemerella anatipestifer, it is a bacterial disease of domestic ducks, geese, turkeys, and other birds. Because of Riemerella anatipestifer wide distribution, high morbidity and mortality, has become one of the important infectious disease endangering the world of duck. The disease has many serotypes, but the disease cross-protection between serotypes are low. Currently, Riemerella anatipestifersis prevention and control mainly rely on antibiotics, but the disease easy to produce drug resistance to antibiotics ineffective and is likely to cause drug residues in food. So that, emphasis on the study of molecular biology, pathopoiesis and immunologic mechanism, will play a very important role in control and prevent the Riemerella anatipestifersis.Sigma is a subunit of bacterial RNA polymerase. It identifies the promoter consensus sequence and transcription initiation with the core enzyme binding. Extracytoplasmic function （ECF） sigma factors are belongs to a70family. It is made of bacteria-mediated adaptive response in face of environmental change. ECF ó factors play an important role in bacteria to adapt environmental stress, invasive and infect the host.There are five genes in the RA-YM genome coding for ECF sigma factors, in order to study the pathogenesis role of ECF a factor in R.anatipestifer strain RA-YM. In this study we use homologous recombination method constructed RA-YM ECFs （RAYM₀3547, RAYM₀0365and RAYM₀8835） gene deletion strains, study its growth characteristics, biochemical characteristics, virulence and growth under environmental stress conditions. The research contents are summarized as follows:1Construction and identification RA-YM ECFs （RAYM₀3547、RAYM00365and RAYM₀8835）Amplified upstream and downstream homology arm of ECFs （RAYM00365, RAYM₀8835and RAYM₀3547） gene use RA-YM genome. Use Overlapping PCR method connected upstream and downstream homology arms and spectinomycin gene together, and then connected it to plasmid pRE112. The recombination plasmids were transformed into the E.coil X7213. In E. coli X7213which contains pRE-ECF as donor strain, RA-YM as receptor strains. After transconjugation, select spectacular resistant colonies on the plate, and then using the PCR method to identify whether there has been exchange of homology arms. In the end, we successfully constructed Riemerella anatipestifer ECFs （RAYM₀3547, RAYM₀0365and RAYM₀8835） gene deletion strains.2The biological characteristics of RA-YM mutant gene deletion strains ECFs gene deletion strains.Comparison growth characteristics and biochemical characteristics of RA-YM ECFs gene deletion mutant strain RA-YMARAYM₀0365, RA-YMARAYM₀8835, RA-YMARAYM₀3574with the parent strain RA-YM, we found that the growth rate of ECFs （RAYM₀3547, RAYM₀0365, RAYM₀8835） gene deletion strains and the parent RA-YM strain are similar in exponential phase. Biochemical experiments found that RA-YM ECFs deletion strains and biochemical characteristics of the parent strain did not change. Comparison growth of RA-YM ECFs gene deletion strains and RA-YM in the environmental stress （acid, bile salts, and H2O2）, found that the three ECFs gene deletion mutants were grow more slowly than RA-YM.3Research the virulence of RA-YM ECFs （RAYM₀3547、 RAYM₀0365and RAYM₀8835） gene deletion strainsThe LD50value of ECFs（ARAYM₀0365, ARAYM08835,△MUYM₀3574） mutant was determined to be approximately3.16x106CFU、3.98x106CFU、5.01×106CFU. The LD50value of RA-YM was determined to be approximately5.01×106CFU. There was not significant different between mutant strains and RA-YM strain. Also, there was not significant different about blood and liver tissue bacterial content. In our study we found that three ECFs gene deletion mutants pathogenicity have no significant change. Speculated the reason may be is that the main role of the ECF which associated with pathogenicity genes, still in RA-YM genome or that RA-YM require multiple ECF participation in collaborative pathogenic. So, next step, we will be construction the RAYM₀4606, RAYM06105single-gene deletion and multi-gene deletion.