bHLH transcription factor SmbHLH92 negatively regulates biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza

Objective:Salvia miltiorrhiza is a valuable herbal medicine with tanshinone and phenolic acid as the main biological active ingredients.The biosynthetic regulation of these bioactive compounds is controlled by a set of transcription factors(TFs).The basic helix-loop-helix(bHLH)transcription factor plays an important role in various physiological and biochemical processes in plants.However,research on bHLH TFs regulating phenolic acid or tanshinone biosynthesis in S.miltiorrhiza is limited.Methods:qRT-PCR was used for gene expression analysis.The subcellular localization of SmbHLH92 was detected by SmbHLHg2-GFP transient transformation into tobacco leaves,and its fluorescence was observed using a confocal laser scanning microscope.The transcriptional activity of SmbHLH92 was confirmed in the AH109 yeast strain.RNA interference hairy roots of SmbHLH92-RNAi transgenic lines were obtained through Agrobacterium-mediated genetic transformation.Ultra performance liquid chromatography(UPLC)was used to detect the changes of phenolic acids and tanshinones.Results:SmbHLH92 is a bHLH transcription factor that is highly expressed in the root and phloem of S.miltiorrhiza.The subcellular localization and transcriptional activity of SmbHLH92 indicated that SmbHLH92 was located in the nucleus and may be a transcription factor.RNA interference(RNAi)of SmbHLH92 in hairy roots of S.miltiorrhiza significantly increased the accumulation of phenolic acid and tanshinone.Quantitative RT-PCR(RT-qPCR)analysis showed the transcription level of genes encoding the key enzymes involved in the phenolic acid and tanshinone biosynthetic pathways was increased in the hairy roots of the SmbHLH92-RNAi transgenic line,comparing with the control line.Conclusion:These data indicate that SmbHLH92 is a negative regulator involved in the regulation of phenolic acid and tanshinone biosynthesis in S.miltiorrhiza.

Cloning and Bioinformatic Analysis of HMGS and HMGR Genes from Panax notoginseng

Objective To clone and analyze 3-hydroxy-3-methylglutaryl coenzyme-A synthase（HMGS） and 3-hydroxy-3-methylglutaryl coenzyme-A reductase（HMGR） genes from Panax notoginseng of four-year old during the flowering period, the key genes involved in the mevalonic acid pathway for saponin biosynthesis. Methods The cDNA sequences of PnHMGS1 and PnHMGR2 were obtained by reverse transcription PCR（RT-PCR） and rapid amplification of cDNA ends（RACE） methods and were analyzed in their secondary structures, subcellular localizations, domains, and the three-dimensional structures of putative proteins by the bioinformatics tools. Fusion genes were constructed by the prokaryotic expression system. Results The two genes were cloned, named as PnHMGS1 and PnHMGR2, respectively, and were both predicted to be located in the chloroplast. PnHMGS1（1410 bp） encoded a predictive unstable protein with 469 amino acids and covered hydroxymethylglutaryl-Co A synthase domain. PnHMGR2（1690 bp） also encoded an unstable protein with 589 amino acids and possessed a hydroxymethylglutaryl-coenzyme A reductase domain and two transmembrane regions. Both of the genes were expressed most in flowers followed by roots, stems, and least in leaves. Conclusion PnHMGS1 and PnHMGR2 are firstly cloned from P.notoginseng as the new member of the HMGR family,and they show the same expression profile as P.ginseng and P.quinquefolius.