| Small regulatory RNAs(sRNAs)are non-coding,short,stranded RNA molecules that play a crucial role in various gene expressions.They are typically 50-500 nucleotides in length and regulate target gene m RNA expression through imperfect base complementary pairing.This regulation of transcription,translation,and other functions of target genes is essential for bacterial adaptation to changing environmental conditions.This study aimed to investigate sRNA expression in Corynebacterium glutamicum under diverse growth conditions,including high salt,acidic environments,DNA damage,heat shock,and peroxide stress.Additionally,the study aimed to create a prospective sRNA database and identify the roles of several putative sRNAs using omics and biochemical techniques.The research involved several primary objectives.Firstly,the study identified the transcription start and stop locations of putative sRNAs using sRNA Detect and integrating with AEPRO bioinformatics techniques.This led to the discovery of 2653 possible sRNAs at various growth stages and 372 potential sRNAs expressed solely under stressful situations.Secondly,the sRNAs were classified into different categories based on their location in the genome,and the genes with potential sRNAs on the antisense chain were analyzed based on their function.Significant differential expression of sRNAs was analyzed based on RNA sequencing data,and a conserved 6C sRNA(sRNA00130)was discovered through homologous comparison.Thirdly,by detecting the expression of potential sRNAs through RT-PCR,24 potential sRNAs with distinct bands were identified.The start site of sRNA02037 transcription was determined to be 1366538 and the end site to be 1366700 through RACE experiments and sequencing methods.Combining the two experimental methods of transcriptome and promoter reporter plasmid,it was detected that sRNA00130 was significantly up-regulated under acid and heat shock conditions.In general,sRNA02037 has a very high expression level,and most stress conditions will lead to the decline of its expression level,which is particularly obvious under the conditions of high osmotic pressure and DNA damage.Fourthly,the overexpression and knockout plasmids of sRNA02037 and sRNA00130 were detected by transcriptome.The deletion of sRNA00130 resulted in expression changes in 105 genes,with 70 genes upregulated and 35 genes downregulated.Multiple genes related to nucleic acid metabolism and DNA repair,including rec N,showed significant differences.Overexpression of sRNA02037 resulted in upregulation of 30 genes and downregulation of 16genes;The deletion of sRNA02037 resulted in a significant upregulation of 96 genes and a significant downregulation of 111 genes.Through direct and indirect effects,sRNAs simultaneously regulate the expression of multiple genes.Finally,preliminary determination of sRNA00103 through direct interaction to regulate the expression of rec N gene was conducted by reporting the plasmid.Site directed mutagenesis of the report plasmid binding site changed the free binding energy of sRNA00130 and rec N-5’UTR region,and the regulatory effect of sRNA00130 on rec N decreased. |
