Analysis And Preliminary Function Of Mycobacterium Tuberculosis SRNA Under Aminoglycosides

Objective In bacteria and other prokaryotic,sRNA is a new regulatory factor that exists in the noncoding region.Its main function is to respond to the external environment stimulating signal by regulatinging gene expression.In recent years,researchers have found that sRNA is associated with antibiotic exposure and development of drug resistance,including aminoglycosides,and less reports in Mycobacterium tuberculosis.Streptomycin is one of the first drugs to be used for anti-tuberculosis treatment,and its similar kanamycin is also used as a second-line drug for clinical tuberculosis treatment.The aim of this study was toscreen the sRNA in response to aminoglycoside stimulation and may be related to the development of drug resistance.The expression of sRNA was detected and the preliminary functional study was carried out.Method Mycobacterium tuberculosis H37 Rv was cultured withthe aminoglycoside streptomycin and kanamycin existed,then the total RNA was extracted to establish three transcriptome libraries.Changes in transcription were analyzed between groups by high-throughput sequencing,and new sRNA werepredicted.By calculating the FPKM values,the expression of sRNA was normalized.In order to screen he sRNA that up-regulated or down-regulated,a hypothesis test was performed on the FPKM values,and the difference was calculated.The expression of sRNA was detected by Northern blot and quantitative real-time PCR.For the further functional analysis of sRNA-ms28,which was co-down-regulated in both streptomycin and kanamycin group,the overexpression of ms28 was obtained by transposing the overexpression plasmid into Mycobacterium tuberculosis H37 Rv,The growth rate and target genes expression was compared with strains electroporated vectors,to analyze the preliminary function.Results Three transcriptome libraries were established using total RNA of streptomycin group,kanamycin group and control group.Differentially expressed genes were selected through differential expression analysis.There are 287(7.2%)genesdifferentially expressed in streptomycin group,of which 184 were up-regulated and 103 were down-regulated.And there are 230(5.8%)differentially expressed in kanamycin group,of which 61 were up-regulated and 169 were down-regulated.Through GO enrichment analysis we found that among all differential coding genes,cell components,membrane proteins and metabolic processes are of the highest degree in the differential gene enrichment.In addition,this study predicted 174 new sRNAs,of which 88 were positive oriented,encoded as ms01-ms88,and the negative orientedsRNAs were encoded as ms89-ms174.Differences in expression between the two groups showed that 5 sRNAs were differentially expressedunder streptomycin.3 of them were up-regulated: ms03,ms75,ms172 and 2 down-regulated: ms28,ms88;6 sRNAs were differentially expressed under kanamycin,2 up: ms113,ms146,4 down: ms28,ms42,ms127,ms137.Small RNA ms28,located in genome 1512822-1513027,is a co-down-regulated sRNA after streptomycin and kanamycin stimulation.Northern blot and q RT-PCR were used to detect the expression of sRNA after streptomycin and kanamycin.7 differentially expressed sRNAs were consistent with the analysis results.The overexpression vector of ms28 was constructed by transforming the shuttle plasmid PMV261,and it was transferred into Mycobacterium tuberculosis to over-express ms28.After phenotypic analysis and q RT-PCR,it was found that ms28 was highly expressed,Six of the potential target genes were up-regulated.Compared with the vector strains,the ms28 overexpression strain has a lefted growth curve and the exponentary phase was 3-4 daysshortened.Conclusion At the level of transcriptome,part of genes expression was changed in Mycobacterium tuberculosis under aminoglycoside exposure,of all the changed genes,genes coding cell,membrane protein and metabolic genes were the most significant.The expression of ms03 and ms172 was up-regulated and ms28 and ms88 were down-regulated under aminoglycoside streptomycin exposure.The expression of ms113 was up-regulated and the expression of ms28,ms42 and ms127 was down-regulated under kanamycin exposure.The overexpression of sRNA-ms28 could make a difference on the growth of Mycobacterium tuberculosis H37 Rv.Irt A,irt B,mbt K,mbt N,FABG2,lpr D are potential target genes for ms28.Ms28 may affect the growth of the target gene by regulating the expression of the target gene,regulating the growth of bacteria and adapting to the external environment.