Screening Of SRNA Chaperone Hfq Target Gene In Salmonella

Salmonella is a Gram-negative,rod shaped bacterium of the Enterobacteriaceae family,which poses a great threat to China's livestock and poultry breeding industry.It can also cause food-borne pollution and seriously threaten human health and food Safety.Bacterial s RNA is a key life activity regulator of bacteria found in recent years.Trans-s RNA requires the bacterial s RNA chaperone Hfq to coordinate the regulation of virulence gene expression,stress response,nutrient absorption,adaptation to environmental changes and quorum sensing.In this study,the Salmonella typhimurium wild type strain was used as a template to construct the Salmonella typhimurium hfq gene deletion strain,and the standard strain and hfq gene deletion strain were cultured to exponential phase and stationary phase respectively,and RNA was extracted for transcriptome.Sequencing,screening differentially expressed genes,performing GO enrichment analysis and Pathway enrichment analysis,and screening some differentially expressed genes for real-time PCR,screening related pathways for in-depth analysis,screening important regulator genes of Salmonella hfq gene deletion strain.1.Construction and identification of hfq gene deletion strain of Salmonella typhimuriumThe hfq gene deletion strain of Salmonella typhimurium was constructed by P22 phage transduction technology.The sequencing results showed that the hfq gene was successfully replaced with the cat resistance gene,indicating that the hfq gene deletion strain of Salmonella typhimurium was successfully constructed.2.Transcriptome analysis of the hfq gene deletion strain of Salmonella typhimuriumThe transcriptome sequencing results of Salmonella typhimurium wild type strain and hfq gene deletion strain were performed by DEseq2 algorithm.In the exponential phase,1055 differentially expressed genes were screened,among which 516 genes were up-regulated and539 genes were down-regulated.In the stationary phase,959 differentially expressed genes were screened,of which 496 were significantly up-regulated and 463 were down-regulated.11 differentially expressed genes such as STM3630,STM2890,STM2874,STM3764,STM4361,STM2898,STM2640,STM2893,STM1583,STM2897 and STM1091 were randomly selected for real-time PCR,and the trend was consistent with transcriptome sequencing,indicating that the transcriptome sequencing results were reliable..The differentially expressed genes of wild type strain and hfq gene deletion strain were annotated in GO database and KEGG database.The exponential phase significant differentially expressed genes were mainly enriched in cobalamin metabolism process and cobalamin biosynthesis process,biological processes such as carbohydrate metabolism,and bacterial chemotaxis,propionic acid metabolism,carbon metabolism,ABC transporter,microbial metabolism in different environments,porphyrin and chlorophyll metabolism,photosynthetic biological carbon fixation,pentose phosphate pathway,Among the 11 signaling pathways in the prokaryotes,such as the carbon sequestration pathway,the citric acid cycle,and the glycolysis pathway.Significant differentially expressed genes in the stationary phase are mainly enriched in the biological processes of cobalamin metabolism,cobalamin biosynthesis,cytoplasmic processes,ribosomal RNA binding processes,porphyrin and chlorophyll metabolism.3.Screening of important co-expression genes of Salmonella typhimurium hfq gene deletion strain based on transcriptome analysisBased on the transcriptome sequencing of Salmonella typhimurium standard strain and Salmonella typhimurium hfq gene deletion strain,the pathogenic mechanism biological process,chemotactic pathway,secretory pathway,ABC transporter pathway,two-component pathway and quorum sensing pathway were screened.Enriched genes of equal signaling pathways.In the exponential phase,the pathogenic pathways,the chemotactic pathway,the secretory pathway,the ABC transporter pathway,the two-component pathway and the quorum sensing pathway were screened for differentially expressed genes,27,12 11,66,68 and 18 genes were screened.In the stable phase,the pathogenic mechanism,chemotaxis,secretion,ABC transporter,two-component system and quorum sensing were screened for differentially expressed genes,36,6,15,49,56 and 18 genes were screened.And Screening of co-expressed genes in exponential phase and stationary phase biological processes and signaling pathways,respectively,in the biological processes of pathogenic mechanisms and in the signaling pathways of the chemotactic pathway,secretory pathway,ABC transporter pathway,two-component pathway and quorum sensing Screening for 14,4,7,20,16 and 5genes,and participating in two or more important co-expressed genes in the above biological processes and signaling pathways are hilA,spaS,invG,spaP,invA,pstS,STM1678,mppA,suggesting that it may be a regulatory target gene for sRNA chaperone Hfq.The results showed that the hfq gene deletion strain of regulates different biological processes and signaling pathways in different periods.In summary,the hfq gene deletion strain was successfully constructed by P22 phage transduction technology.The wild type strain and hfq gene deletion strain were cultured to exponential phase and stable phase for transcriptome sequencing and screening.Differentially expressed genes were analyzed by GO enrichment analysis and Pathway enrichment analysis.The differentially expressed genes of functional biological processes and signaling pathways in the exponential phase and stationary phase were screened,and 8 possible target genes of hfq were found.