Screening Of SRNA GcvB Regulatory Genes In Salmonella

In order to study the effects of small RNA GcvB(small RNA,sRNA)on the survival,virulence and pathogenicity of Salmonella,the target genes that GcvB may regulate were screened.This study used high-throughput transcriptome sequencing(RNA-seq)technology to compare the mRNA of Salmonella LT2 Wild-type(WT)strain and Salmonella LT2 gcvB gene deletion strain,and comprehensively predict the number and type of GcvB regulatory genes,using GO and KEGG analyses.The database was analyzed by informatics,and the interaction site was predicted by Target RNA2.Fluorescence quantitative PCR was performed on the Top10 diference genes,ABC transporter pathway genes and soft ware Target RNA2 predicted partial genes.Further analysis of the main functions of the screened genes and the biological processes involved.The results are as follows:1.Identification and growth curve determination of Salmonella gcvB gene deletion strainThe Salmonella LT2 WT strain and the Salmonella LT2 gcvB gene deletion strain were identified by PCR.The results showed that the gcvB gene sequence was replaced by the cat gene sequence,and the small RNA GcvB was knocked out.The growth curve of the two strains was determined by turbidimetry.The results showed that the Salmonella LT2 WT strain entered the Exponential phase at 6 h,entered the log phase at 3 h of Salmonella LT2 gcvB deletion,and the two strains entered the stationary phase at 12 h.indicating that the growth and metabolism of bacteria after the loss of small RNA GcvB is affected.2.Transcriptome sequencing analysis of Salmonella gcvB gene deletion strainThe transcriptome sequencing analysis of Salmonella LT2 WT strain and Salmonella LT2 gcvB gene deletion strain was carried out by RNA-seq technology.The results showed that there were 1244 differentially expressed genes,of which 678 were up-regulated and 566 were down-regulated.Top10 differentially expressed up-regulated genes: STM1131,sicA,pduD,pduC,prgK,down-regulated genes: nirB,narG,napF,napD,STM2344,3116non-significant difference genes.The KEGG and GO data analysis methods were used toanalyze the sequencing data.The KEGG database analysis showed that the differentially expressed genes were mainly involved in 14 kinds of signal pathways such as bacterial chemotaxis,microbial metabolism in different environments,two-component system and methane metabolism,The results of databace GO showed that the differentially expressed genes mainly involved the cellular components of bacterial flagella,redox activity,iron ion binding and other molecular functions,cell respiration,and cobalamin metabolism processes.The GO and KEGG data analysis methods were used to analyze 13 genes in the ABC transporter pathway and 7 genes predicted by Target RNA2.The results showed that the gene involved the manganese/iron transport system permease protein and the arginine transport system substrate binding protein.Such as signal pathways,cytoplasm,nitrate and other cellular components,metal ion binding,polyamine binding and other molecular functions,anaerobic respiration,amino acid transport and other biological processes.3.Salmonella sRNA GcvB regulatory gene screeningThe top 10 differentially expressed genes,13 genes in the ABC transporter pathway,and7 genes predicted by Target RNA2 were verified by real-time PCR.The results showed that the up-and-down regulation of 30 genes was consistent with the results of RNA-seq sequencing.The above results indicate that under the condition of knocking out small RNA GcvB,the growth and metabolism of Salmonella have undergone complex changes.Based on the sequencing of RNA-seq transcriptome,the possible regulatory genes of small RNA GcvB were verified by real-time PCR.According to the results,the foundation is established to further explore the interaction between small RNA GcvB and regulatory genes and the regulation mechanism of small RNA and the pathogenic mechanism of Salmonella.