Study On Biological Characteristics Of APEC Modified By Noncoding Small RNA (RyhB) And Screening Of Its Differential Proteins

Avian pathogenic Escherichia coli(APEC)is a bacterium that can cause local or systemic infectious diseases in poultry and can be associated with many viral and bacterial diseases.With the development of the world intensive chicken industry,the incidence and mortality of E.coli disease has gradually increased,causing significant losses to the development of poultry industry.In the study of Escherichia coli,small non-coding RNA(s RNA),a novel gene regulator,which regulates the expression of a level-modulating gene after transcription,mainly involves in these regulatory pathway,including regulation of transcription and regulation,iron metabolism pathway,glucose metabolism pathway,membrane protein biosynthesis,population induction system and pathogenicity regulation of pathogens.Ryh B is also a small RNA,which plays a key role in the pathogenesis of bacterial disease.In addition,Ryh B can participate in the regulation of iron metabolism.In Vibrio cholerae and Shigella dysenteriae,Ryh B was found to regulate bacterial dynamics,chemical chemotaxis,biofilm,acid tolerance,redox or even virulence genes.Therefore,studying the link between Ryh B and APEC can establish experimental basis.for preventing and controlling APEC.In this study,the effects of RyhB deficiency on the biological characteristics of bacteria were explored.Proteomics were used to compare the differentially expressed proteins in wild and deficient mutant,among those APEC toxicity were screened.The relationship between Ryh B and APEC pathogenicity was further revealed.The specific research contents and results are as follows:1 s RNA-Ryh B on the biological characteristics of avian pathogenic Escherichia coliIn this experiment,we studied the effects of APEC RyhB including the growth curve assay,the experiment of tissue culture assay,the observation of the micro-structure of the cell,the biofilm formation ability assay,the LPS integrity assay and the environmental tolerance test on the Biological Characteristics of Bacteria.The results of growth curve showed that Ryh B did not affect its growth performance.The results showed that the amount of bacteria injected into △Ryh B liver tissue was 6.91 times higher than that of wild strain.The ultrastructural observation showed that,with same growth conditions,the parent strain AE17 had a complete fimbriae,and the △Ryh B strain lost complete fimbriae;biofilm assay results showed that,deletion of Ryh B weakened the biofilm formation ability,the complementary strain has been restored,but LPS integrity assay results showed that the deletion of Ryh B did not affect the integrity of LPS;environmental tolerance test showed that the △Ryh B was not sensitive to oxidation;heat stress and acid resistance enhanced;alkali resistance weakened.2 Target proteins in Avian pathogenic Escherichia coli regulated by s RNA-Ryh B were screenedTo explore Ryh B may control the virulence genes.Proteomic techniques were used to compare the expression of differential proteins between wild and deficient strains from the protein level.The results showed that 62 differential proteins were found in comparison with wild strain.Among them,Omsc,Galu,Nlpe,Tata,Gmh A,pro X,in FA,cys E may be Ryh B directly controlled virulence-related genes.The results of GO(Gene Ontology)showed that the differentially expressed proteins were cell membrane,extracellular domain,organelle,and nuclear material.The molecular function mainly showed antioxidant function,protein binding,catalytic activity,molecular function regulation,signal transduction,transport protein function,etc.;in the biological process,including regulation of biological regulation,movement process,metabolic process,multicellular biological processes,stress response,a single organism adjustment process.The transcription level of 10 differential protein genes was detected by real-time fluorescence quantitative PCR.The results were consistent with proteomics.3 s RNA-Ryh B regulation of avian pathogenic Escherichia coli acid tolerance and the impact of biofilm formation target protein analysisIn this study,the protein related to acid tolerance was screened by proteomics,and the transcription level of the selected differential protein gene was detected by real-time fluorescence quantitative PCR.The results were consistent with proteomics.On the basis of the selected differential proteins,the on-line prediction software was used to screen the target proteins which may be directly related to the biofilm formation.The expression vector p ET28a-cys E was successfully constructed and the soluble cys E protein was obtained.The recombinant plasmid was detected by Western Blot,there was a protein band at about 36 k Da,which indicated that the expressed product had good specificity.The results of the above studies show that the deletion of RyhB can affect the expression of avian pathogenic E.coli fimbriae and reduce the amount of bacteria in the liver,supress the formation of biofilm,enhance heat stress,enhance acid resistance and weaken alkali resistance.Which indicated that Ryh B played an important role in the regulation of APEC virulence.Combined with proteomics technology to screen out proteins related to biofilm formation and acid tolerance.This study laid a foundation for the further study of Ryh B regulatory target and also provided the experimental basis for further study of the pathogenicity of APEC and the mechanism of host resistance.