反义RNA介导的大肠杆菌非必需基因rpsF的基因沉默

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

摘要:

摘要:【目的】探讨反义RNA 技术介导的大肠杆菌非必需基因rpsF基因沉默导致菌体生长受抑制的原因。【方法】将rpsF基因5' 端41－230 bp的片段反向插入带有末端配对结构的反义表达载体pHN678，获得重组质粒，导入大肠杆菌宿主获得反义RNA菌株Escherichia.coli/pHNF，并用诱导剂IPTG诱导反义RNA表达，通过与对照菌E.coli/pHN678 的液体生长状态差异判断菌体生长表型;采用Real time RT-PCR方法跟踪分析转录水平。【结果】构建了针对rpsF的反义RNA菌株，且其生长受抑制程度与IPTG浓度呈正相关。IPTG浓度为100 μmol/L时，菌体生长未受抑制，但靶基因rpsF的mRNA量降低了36%，而rpsR是位于同一操纵子下游的必需基因，其转录水平却未受影响;IPTG浓度为200 μmol/L时，菌体生长明显受抑制，经分析发现rpsR转录水平降低了12%。【结论】反义RNA菌株E.coli/pHNF 生长受抑制的原因是由于此反义RNA引起了同一操纵子下另一必需基因rpsR的转录水平降低。

关键词:关键词:反义RNA，非必需基因rpsF，基因沉默，操纵子，Real time RT-PCR

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.

Key words:Keywords: antisense RNA，nonessential gene rpsF，gene silencing，operon，real time RT-PCR