The Effects Of Small Non-Coding RNA (RyhB) Deletion On The Avian Pathogenic Escherichia Coli Pathogenicity

The Avian pathogenic Escherichia coli can cause avian local or systemic acute and chronic infection,due to the complexity of its virulence factors,it brought serious harmful to the development of the poultry industry.In recent years,The non-coding small RNA which was proposed as a newly discovered gene regulator,the post transcriptionally can regulate the level of gene expression.The Escherichia coli as RNA mediated regulation of pathogenic bacteria,it has been conducted in-depth study about its mechanism of virulence,pathogenic mechanism and metabolism.The Ryhb is one of the regulation target mRNA has the highest number of small RNA molecules,it is a key part of the Escherichia coli many regulatory networks and it plays a important role in the pathogenesis of bacteria.Therefore,it has significant effects to study the relationship between RyhB and APEC pathogenicity.Taking sRNA(RyhB)as the object of study,this research constructed APEC deletion strain △RyhB and the complemented strain△RyhB-comp to study the effects of gene deletion on the biological characteristics,virulence factor transcription level,adhesion of the CEF cells,half lethal dose and pathological changes of chickens infected with ring,it revealed the relationship between ryhb gene and APEC pathogenicity and it will lay foundations for further research on APEC adhesion and invasion of host cell pathogenic function regulation pathway(pathway).The concrete contents and results are as follows:1.The construction of RyhB deletion strain and the complemented strain on avian pathogenic Escherichia coli.The experiment used the red homologous recombination technology,and amplified RyhB-Up,PKD3-Cm and RyhB-Down by PCR.Using the overlap method and amplified RyhB homologous recombination fragment for shooting the target sequence.According to 30 degree heat induced protein,the homologous recombination fragment is electroporated into the PKD46 containing original strain,it carried out target gene replacement.The positive transformation was selected from the chloramphenicol resistance marker and PCP20 was transferred to positive strains to eliminate chloramphenicol resistance,It is successfully constructed by APEC wild strains of RyhB deletion strain] △RyhB by PCR verification.Synchronization by RyhB gene amplification,The obtained RyhB fragment and pSTV28 vector were digested with EcoR Ⅰand Hind Ⅲ respectively,connecting with T4 DNA ligase and the connect products were transformed into Escherichia coliDH5α alpha competent cells,Selection of positive clone by chloramphenicol resistant plate.PSTV28-RyhB plasmid DNA was transferred into △RyhB by electroporation and selected of CM resistance to make sure get a complemented strain △RyhB-comp by PCR verification.2.Evaluation of the biological characteristics of RyhB deletion strain and the complemented strain on avian pathogenic Escherichia coliThe experiment studied the difference of the biological characteristics of avian pathogenic Escherichia coli RyhB deletion by studying the growth curve,motion performance,reactive oxygen species(ROS)inhibition and avian β-defense6 tolerance test respectively.The results showed that The APEC deletion strain △RyhB and complemented strain△ RyhB-comp had no significant effect on the growth performance;The deletion of RyhB can reduce the AE17 motion performance significantly,and constructs the reply plasmid △RyhB-comp motion performance has returned;three strains on active oxygen pressure had no significant difference,indicating that the deletion of RyhB can not affect the tolerance of APEC to reactive oxygen species;Compared with AE17,the avian β-defensin 6 tolerance of △RyhB decreased significantly,and the strain △RyhB-comp tolerance has returned.3.Analysis on the effect of RyhB deletion strain and the complemented strain on avian pathogenic Escherichia coli Virulence and pathogenicity.By the detection of virulence gene transcription,CEF cell adhesion,LD50 and the pathological tissue of chicken change,the study is to explore the effects of deletion of the APEC RyhB on the virulence and pathogenicity.The results showed that the transcription of bcfA,fyu A,tsh,luxS,fim C,ompA genes of △RyhB was significantly decreased(P<0.01)and its mRNA transcription levels was decreased by19%,52%,20%,14%,28% and 52% respectively while the iss and ibeA genes were increased about 1.14 folds and 1.2 folds respectively.The adherence of △RyhB was decreased by 62.5% compared to AE17 and the △RyhB-comp was increased about 1.4folds compared to △RyhB.The LD50 results show that compared to the AE17,△ryhb virulence decreased about 143.5 folds and compared to △ryhb,△RyhB-comp virulence recovery.Chicks pathological slice observation shows that the APEC can cause varying degrees of necrosis,degeneration of chicken heart,liver,spleen,small intestine tissue,accompanied by congestion and congestion.The experiment successfully constructed the APEC deletion strain △RyhB and complemented strain△RyhB-comp.Research results show that,the deletion of RyhBcan make the performance of APEC reduced.,avian β-defense 6 tolerance decreased,the expression level of virulence genes decreased,The adhesion of CEF cells decreased and LD50 levels of decline.It is suggested that RyhB has a very important regulatory effect on the virulence and pathogenicity of APEC and it will provide some theoretical reference for further research on the pathogenesis of APEC by RyhB.