关键词: 沙门菌, λ-Red同源重组系统, GcvB, 伴侣蛋白Hfq, oppA

Abstract: To explore the possible binding sites of Hfq on GcvB, in this study, the Hfq binding preference U-rich motif in GcvB was screened. The mutant and truncated strains of the GcvB U-rich motif were constructed by the λ-Red homologous recombinase system, and the GcvB terminator stem extension strain was constructed at the same time. P22 phage transduction technology was used to construct corresponding hfq gene deletion strains and oppA::lacZ gene fusion strains. The gcvB gene transcription expression level of the recombinant strain was detected by qRT-PCR, and the oppA gene protein expression level was detected by the β-galactosidase test. The results showed that the mutation of U₅ motif did not cause obvious changes in the transcription and expression level of gcvB gene, but when the U₈ motif was truncated to U₅ and U₄, it was reduced by 40.5% and 37.5%, respectively. After the GcvB terminator stem of the U₄ truncated strain was extended, it was discovered that the transcription and expression level of gcvB gene was restored,but the ability to Hfq negatively regulating oppA mRNA was weakened after co-transcription. The above results show that the U₅ motif has no obvious relationship with Hfq maintaining the stability of GcvB,but the U₈ motif is important for Hfq to assist GcvB with regulating oppA mRNA negatively after co-transcription and maintain GcvB stability. In summary, we speculate that the U₈ motif is the binding site of Hfq on GcvB.

Key words: Salmonella, λ-Red homologous recombination system, GcvB, chaperone Hfq, oppA