| Dan shen(Salvia miltiorrhiza Bunge)is a traditional Chinese herb with a long history.Due to its complete genome sequencing,short growth cycle,and great adaptability,Danshen has been an important model plant.With bitter tasty and slightly cold,Danshen can activate blood stasis,eliminate heat,and cool blood.Clinically,it was used to treat cardiovascular,cerebrovascular,diabetes,and other diseases,which is of extremely high medicinal value.Phenolic acids and tanshinones are two of the most important active medicine components.It’s an effective strategy for exploring the diversification of sources of medicine to research secondary metabolites in S.miltiorrhiza.Basic-helix-loop-helix(bHLH)family transcription factors can work in growth and development,secondary metabolism,stress tolerance,and many physiological processes.SmbHLH124 and SmbHLH92 have been explored in their biological functions by this research group,which reported that the hairy root knocking out SmbHLH124 completely repressed the growth of hairy roots.To further understand the biological function of bHLH transcription factors,the functional study of SmbHLH125,which belongs to the N subfamily with SmbHLH124,was achieved with the following results:1.The SmbHLH125 gene is successfully cloned from S.miltiorrhiza,containing a279 bp ORF that encodes 93 amino acids.It belongs to bHLH super family.Based on characterization analysis,SmbHLH125 is highly homologous to ATBS1,located in the nucleus and is highly expressed in flowers and roots.2.SmbHLH125 impacts the growth and development of S.miltiorrhiza.The growth of OE-SmbHLH125 roots is completely repressed.The growth of KO-SmbHLH125 roots is no significant difference compared with control.3.SmbHLH125 multiply regulates the secondary metabolic pathways in S.miltiorrhiza.HPLC shows that the content of rosmarinic acid(RA)is notably decreased in mutants,while the content of salvianolic acid B(SAB)and four kinds of tanshinones,including tanshinone I,cryptotanshinone,dihydrotanshinone,and tanshinone IIA,are significantly increased.(1)Transcriptome results shows that the expression of Sm4CL3 and Sm4CL7 are upregulated and that of Sm4CL9 is downregulated.They are involved in the formation of coenzyme A esters of hydroxycinnamic acid and its derivatives in phenolic acid synthesis.Combine with the analysis of Y1 H assays,SmbHLH125 binds to Sm4CL3 to negatively regulate the expression of the gene to regulate phenolic acids biosynthesis.(2)In tanshinones biosynthesis,SmbHLH125 directly binds to G-box elements of promoters to SmDXR,SmAACT2,SmMK,SmIDI,and SmCPS to positively regulate the expression of genes,and negatively regulate SmHMGR1 and SmKSL to regulate tanshinone biosynthesis.(3)Integration of transcriptome and metabolome analysis,it is found that the expression of multiple enzyme genes and metabolites in the biosynthesis of phenolic acids and Terpenoids is changed.Multiple CYPs have a strong positive correlation with tanshinones and phenolic acids,SmLAC7 and SmLAC27 have a strong positive correlation with trimers and tetramers included Salvianolic acid B.4.SmbHLH125 caused the expression of a variety of transcription factors to change.Transcriptome results showed the expression of various types of transcrip tion factors changed,including bHLH,MYB,AP2/ERF,NAC,and so on.New G ene(New Gene＿13182)、AP2/ERF member At1g50680、HD-Zip member REVOLUT A-like is of the highest expression level.SmROC-like、ANTHOCYANINLESS 2and WRKY26 are of the lowest expression level.In summary,SmbHLH125 is cloned and characterized.Gene function research indicates SmbHLH125 affects various of genes and metabolites of phenolic acids and tanshinones biosynthesis,modulating secondary metabolism process of S.miltiorrhiza.It also affects the expression levels of multiple transcription factors.Finally,combined with transcriptome and metabolome,the possible interaction relationship between CYP450 and laccase and phenolic acids and tanshinones is predicted.Our study initially demonstrated the regulatory network of SmbHLH125 in secondary metabolites synthesis in S.miltiorrhiza. |
