| Danshen（Salvia miltiorrhiza）is a valuable and time-honored traditional Chinese herbal medicine that is an effective medicine for the prevention and treatment of cardiovascular disease,of which phenolic acids and tanshinones are the main medicinal active ingredients.As a medicinal model plant,the metabolism of the active ingredients is one of the hot topics in the current research.However,the transcriptional regulation of the biosynthesis of phenolic acids and tanshinones is currently unclear.Hairy roots with SmbHLH92 overexpression and knockout by CRISPR-Cas9 were obtained in an Agrobacterium-mediated system.The thesis revealed that SmbHLH92 plays a dual regulatory role in the biosynthesis of phenolic acids and tanshinones in hairy roots of Salvia miltiorrhiza by HPLC.To further analyze the role and mechanism of SmbHLH92 in the secondary metabolic network of S.miltiorrhiza,the combined analysis of transcriptome and metabolome and Yeast one-hybrid（Y1H）assay were then carried out.The main results are as follows:1.Based on the genome and transcriptome database of S.miltiorrhiza,a bHLH transcription factor SmbHLH92 was identified,and mainly expressed in the root.SmbHLH92 had 1303 bp in total length with 3 exons and 2 introns encoding a protein with 226 amino acids.The calculated molecular mass of the protein was 25.39 k D,and p I was 6.31.SmbHLH92 transcription factor had a conservative bHLH domain and is localized in the nucleus.2.SmbHLH92 had a promoting effect on the growth of hairy roots.Compared with the control,overexpression of SmbHLH92 increased the biomass of hairy roots significantly,of which fresh weight increased by 1.76-1.82 folds and dry weight increased by 1.43-1.56 folds,respectively.The fresh weight and dry weight of SmbHLH92-KO roots did not change significantly compared with the control roots.3.SmbHLH92 had a dual regulatory effect on phenolic acids and tanshinones.Based on HPLC analysis,SmbHLH92 positively regulated Rosmarinic acid,but negatively regulated Dihydrotanthionone I and Cryptostanshinone.Regardless of the changes of SmbHLH92 expression level,the accumulation of Salvianolic acid B was inhibited,while the accumulation of Tanshinone I and Tanshinone IIA were promoted.4.SmbHLH92 had a dual regulatory effect on the expression of key enzyme genes in the biosynthetic pathways of phenolic acids and tanshinones.Transcriptome analysis found that overexpression of SmbHLH92 could significantly upregulate the expression levels of Sm4CL6,Sm4CL8 and Sm RAS5,but significantly downregulate the expression levels of Sm RAS in the biosynthetic pathway of phenolic acid.In the tanshinone biosynthetic pathway,the expression levels of Sm HMGR3 and Sm HMGR4 were significantly upregulated while the expression levels of Sm DXS3,Sm MDC and Sm MCT were significantly downregulated.5.Yeast single hybridization assay showed that SmbHLH92 could bind to G-box on the promoters of Sm HDS1,Sm CPR,Sm CPS4,Sm DXS5,Sm MCS,Sm GPPS.LSU,Sm DXR,Sm HDR2,Sm HMGR4,Sm GPPS.SUII.2,Sm AACT2.Combination with transcriptome results revealed that SmbHLH92 might regulate the biosynthesis of tanshinone compounds by directly binding to the G-boxes of Sm HMGR4,Sm GPPS.LSU,Sm GPPS.SUII.2 and Sm DXR promoter. |
