The Construction And Application Of Antisense RNA Mediated Gene Silencing Of Ribosomal Protein Genes In Escherichia Coli

The discovery of antibiotics is the miracle to modern medical science.However,the abuse and unreasonable application of antibiotics have triggered the appearance of drug-resistant bacteria.Therefore,developing new method and technology in antimicrobial drug development area is an important problem for the moment.This study applicated antisense RNA gene silencing technique,selected 45 E.coli ribosomal protein genes as the target,successfully constructed 45 antisense RNA expression strains,and some partially constructed strains were used in two parts,one was to analyse the susceptibility of clinical constantly usage antibiotics,the other was to screen the new antibacterial drugs from the fermentation samples of Actinomycetes.1.Forty-five genes encoding ribosomal proteins from E.coli were selected to design the antisense fragments and plasmid pHN678 was used as the antisense RNA expression vector to construct recombinant plasmids.Then the recombinant plasmids were transformed into competent cell of Escherichia coli DH5a,respectively.Twenty-four IPTG-inducing antisense RNA strains targeted 50S subunit of ribosomal proteins,and 21 IPTG-inducing antisense RNA strains targeted 30S small subunit of ribosomal protein were obtained.2.This study also analysed antisense RNA gene silencing efficiency of all the antisense RNA strains through growth phenotype assay and molecular biology method.Through growth phenotype assay,thirty-four antisense RNA strains targeted essential genes for growth,nine antisense RNA strains targeted nonessential gene for growth showed growth inhibition phenomenon.Two antisense RNA strains targeted growth nonessential gene did not show growth inhibition phenomenon.Through molecular biology method,we validated that antisense RNA fragment can bind to mRNA of target gene resulting in silencing the target gene.3.The silencing efficiency of E.coli DH5a/rpsF antisense RNA strain in transcripion level was validated through Real time RT-PCR.We found that silencing of nonessential gene can decrease its mRNA level of downstream essential gene,and lead to the growth inhibition.4.Twenty-nine antisense RNA strains were used to analyse the sensitivity of 20 antibiotics.We found that antisense RNA strains were more sensitive to specific and synergistic antibiotics.parts of strains were also resistant to antibiotics.5.Seven antisense RNA strains which targeted rpsA,rpsL,rpsR,rplC,rplS,rplT.rpmA,respectively,were used to screen active compounds from sixteen hundred Actinomyces fermentation rough extracts.The positive rate were 1.44%,1.69%,1.25%,1.19%,1.50%,1.25%,and 1.50%,separately for primary screening and 1.19%,1.50%,1.00%,0.88%,1.38%,1.06%,1.44%respectively for the secondary screening.