Antisense RNA mediated gene silencing of nonessential gene rpsF in Escherichia coli

Abstract:

Abstract:［Objective］ We explored the cause of cell growth inhibition by antisense RNA mediated nonessential gene silencing of rpsF gene in Escherichia coli．［Methods］The 41－230 bp fragment around 5' end of gene rpsF was reversely cloned into antisense expression vector pHN678，which is flanked with a paired-termini．The recombinant plasmid was named pHNF．Then it was transformed into E.coli to produce antisense RNA strain E.coli/pHNF．Antisense RNA expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG)，the difference of liquid growth phenotype was identified between E.coli/pHNF and the control strain E.coli/pHN678; and gene transcriptional level was measured by Real time RT-PCR.［Results］We obtained one antisense RNA strain targeted rpsF．We found that the reduced growth rate of this strain was positively related to the IPTG concentration． When IPTG was 100 μmol/L，the cell growth was not inhibited whereas the mRNA amount of rpsF had decreased by 36%，and mRNA of essential gene rpsR in the same operon did not decayed．However，when IPTG reached 200 μmol/L，the cell growth was obviously inhibited and rpsR mRNA was reduced by 12%.［Conclusion］ The essential gene transcription level of rpsR decreases with the nonessential gene silencing of rpsF in the same operon，and leads to the growth inhibition of E.coli/pHNF.