RÉSUMÉ
Objective:To investigate the mechanism of Mycobacterium tuberculosis ( Mtb) WhiB2 in the pathogenesis of tuberculosis. Methods:A recombinant vector of pET28a-WhiB2 for heterogeneous expression of WhiB2 was constructed. The target protein WhiB2 and the inclusion bodies were purified. The differences between denatured and non-denatured WhiB2 were analyzed by circular dichroism and nuclear magnetic resonance. Ferrous ion (Fe 2+ ) was used to restore the iron-sulfur cluster of WhiB2. The interaction between WhiB2 and the upstream promoter sequence of the WhiBMtb gene was analyzed by nuclear magnetic resonance. The tertiary structure of WhiB2 and interacting proteins were analyzed and protein structure alignment was performed based on bioinformatics. Results:The structure of the renatured WhiB2 was basically the same as that of the non-denatuous WhiB2. In addition, Fe 2+ could restore the iron-sulfur cluster of WhiB2. It was found that WhiB2 could bind to the upstream promoter sequence of the WhiBMtb/WhiB2Ms gene. Conclusions:Mtb WhiB2 played a key role in the pathogenesis of tuberculosis, which would contribute to future exploration of novel targets against Mtb.
RÉSUMÉ
Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.