The Cloning,Expression And Biological Analysis Of Repressor Protein ArgR In Corynebacterium Glutamicum ATCC 14067

L-arginine has been proved to be a precursor of nitric oxide(NO),and the NO molecular has many functions such as relaxing and dilating blood vessels.Therefore,L-arginine can be used in many clinical fields and has received increasing attention in recent years.Corynebacterium glutamicum ATCC 14067 is a spore-free gram-positive bacterium,and it is an important producer of various amino acids(including arginine).ArgR is an important regulatory protein in the arginine biosynthesis pathway in Corynebacterium glutamicum,but its regulatory mechanism is not yet fully understood.Currently,genome-wide sequencing of Corynebacterium glutamicum ATCC 14067 had been completed.Based on these data,transcriptome results from RNA-seq can be used for quantitative and functional association analysis of ArgR.The results obtained were verified by electrophoretic mobility shift assays.The specific research content and results can be summarized as follows:(i)The cloning and expression of ArgR from C.glutamicum ATCC 14067.The recombinant strain E.coli BL21(DE3)/pET22b-arg/R was constructed and induced to express ArgR protein.The analysis of ArgR protein using Native PAGE and molecular sieve columns showed that the relative molecular weight of ArgR protein is about 108 kDa,and the ArgR protein is a hexameric structure in its native state.(ii)The preliminary analysis of the biological functions of ArgR using RNA-seq technology.Gene expression in the wild-type strain C.glutamicum ATCC 14067 was compared to that in the argR overexpression strain C.glutamicum ATCC 14067/pEC-XK99E-arg/R and the ArgR function loss mutant C.glutamicum ATCC 14067 AargR.The maximal gene expression difference was found between C.glutamicum/XK99E and C.glutamicum AargR.There were a total of 487 differentially expressed genes,among which the expression of the seven enzymes located in the arginine operon argCJBDFRGH had the largest difference.The expression levels of carbamyl phosphate synthetase genes car A and carB also differ greatly.These results may indicate that the loss of function of ArgR relieves the repression of these genes,indicating that the expression of arginine biosynthesis genes may be regulated by ArgR.(iii)Using the electrophoretic mobility shift assays to verify the regulation of ArgR on arginine biosynthesis genes.The results showed that ArgR protein can bind to argC,argB,argG,and car A gene fragments in vitro and had the highest degree of binding to argB gene,indicating that ArgR protein may regulate the transcription of related genes through binding to these target sites.