The Research On PhoP/Q Regulate Virulence Gene In APEC And Response Gene Of Intestines Were Infected By APEC And PhoP/Q Mutant

Avian pathogenic Escherichia coli(APEC)virulence factors of pathogenic are complex.APEC infected chick intestines by endotoxin(LPS),destruction of the epithelial tissue integrity and cell structure,the induced defensive response gene expression.PhoP/Q two-component system is a signal transduction regulation system and it regulat bacterial virulence and many cell activity in Gram-negative bacteria.PhoP/Q two-component system plays an important role in the pathogenicity of bacteria.This experiment we studied impact on the biological characteristics and virulence of PhoP / Q gene mutant in APEC.In this study,gene microarray technology is applied for screening the differentially expressed genes of wild strains and mutant strains screening PhoP may regulate virulence genes.In this study.RNA-Seq technology is applied for screening the differentially expressed genes of chick small intestine which was infected by APEC and mutant strains.Further revealed the relation between PhoP/Q and pathogenicity of APEC.The specific contents and results are as follows:1.PhoP/Q gene deletion on avian pathogenic Escherichia coli biological characteristics and virulence effects.Observation of the ultrastructure in bacteria,hydrogen peroxide sensitivity experiments,serum bactericidal and microbial load organization experiment were used to explore of PhoP/Q two-component system of gene mutant effects on biochemical characteristics and virulence of APEC.The results show that under the condition of the same culture,AE17 can express the complete fimbriae,phoP and phoQ single gene mutant and phoP/Q gene mutant can not can express the complete fimbriae.The results of hydrogen peroxide Sensitivity is not obvious.The results of serum bactericidal experiment show that compared with the AE17,gene mutant serum bactericidal is not sensitive.The results of Microbial load tissues show that Roman chicken liver and spleen were infected AE17 bacteria microbial loads were1.72×10~7CFU /g and 2.23×10~7CFU/g,however,Roman chicken liver and spleen were infected PhoP/Q microbial load of the bacteria were 1.37×10~5 CFU/gand 4.23×10~6 CFU/g.liver and spleen of bacteria microbial loads were reduced in gene mutant.2.Gene microarray technology is applied for screening PhoP/Q regulation of virulence genes.In order to explore PhoP/Q two-component system impact on avian pathogenic escherichia coli genome expression spectrum screened out may be regulation of virulence genes by PhoP.Using DNA microarrays and quantitative reverse transcription-PCR analyses differentially expressed genes.The results show that there were 292 genes which were different multiples more than two times,170 were up—regulated genes and 122 were down 一 regulated genes.GO analysis results revealed that the differentially expressed genes were related to biological process,metabolism,cell,cell membrane,combination,catalytic activity,electronic carrier activity,enzyme activity,receptor activity,structure,molecular activity,transcriptional regulation,transshipment activities.7 differentially expressed genes were verified with real time quantitative PCR.3.Application of RNA-Seq technology for screening differentially expressed genes in chicken small intestine cells affected by APEC and phoP/Q gene mutantIn order to screen the differentially expressed genes of chick small intestine which was infected by APEC.In this study,APEC strains were used to injected two weeks of chicks of leg muscle,the control group were injected physiological saline to leg muscle,select lesions serious of gut and the control group of gut.In this study.RNA-Seq technology is applied for screening the differentially expressed genes of chick small intestine which was infected by APEC,The differentially expressed genes were analyzed with gene ontology(GO)and KEGG to screen the significant signaling pathway.Several differentially expressed genes were verified with real time quantitative PCR.The results show that there were 131 genes which were different multiples more than two times,74 were up—regulated genes and 57 were down 一 regulated genes.GO analysis results revealed that the differentially expressed genes were related to protein binding,immune response,transporter activity,etc.KEGG analysis results revealed that the differentially expressed genes were related to PPAR,metabolism,Cytokine-cytokine receptor interaction,Jak-STAT,RIG-I-like receptor,phagosome signaling pathways,etc.The results show that there were 172 genes which were different multiples more than two times,93 were up—regulated genes and 79 were down 一 regulated genes.GO analysis resultsrevealed that the differentially expressed genes were related to REDOX activity,transshipment,metabolic processes,immune response,etc.KEGG analysis results revealed that the differentially expressed genes were related to MAPK signaling pathways,sticky spot,PPAR,phagosome,metabolic pathways,etc.The research results show that gene mutant can not express the complete fimbriae.Liver and spleen of bacteria microbial loads were reduced in gene mutant.PhoP/Q two-component system plays an important role in APEC virulence.Gene microarray technology is applied for screening differentially expressed genes and bioinformatics analysis and literature reports,screen crcA,ompT,yrbL,ybjx,rstA,rstB,fliA hslJ and degP may direct regulation of virulence genes by PhoP.RNA-Seq technology is applied for screening MAdCAM-1,S100A9,CCL19,CCLI10,DDX3 X,LEAP2,CALB1,SOCS3 and MT4 immune response differentially expressed genes,This study laid a foundation for the further study of the pathogenic of APEC and the host disease resistance mechanism.