Molecular Mechanism Of SmbHLH74 In Regulation Of Tanshinone Biosynthesis In Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge,a traditional Chinese medicine,is famous for its significant effect on the prevention and treatment of cardiovascular and cerebrovascular diseases.To meet the continuously increasing market demand,the quality of this herb has becoming the research focus.Tanshinones and salvianolic acids are main active ingredients of Salvia miltiorrhiza.To reveal the metabolic regulation network of such ingredients is of significance for improving the quality of Salvia miltiorrhiza.Transcription factors play an important role in regulating plant secondary metabolism.In this study,the function of Salvia miltiorrhiza transcription factor SmbHLH74(which was mainly and highly expressed in roots)in the regulation of tanshinone biosynthesis was investigated.Overexpression of SmbHLH74 significantly inhibited the accumulation of tanshinones.The molecular mechanism of its inhibition effect was analyzed by electrophoretic mobility shift assay,chromatin immunoprecipitation and dual luciferase reporter system.Further,SmMYC2,the master switch of JA signaling,was demonstrated as a direct upstream transcription factor to repress the expression of SmbHLH74.The main results were as follows:1.SmbHLH74 gene was cloned from Salvia miltiorrhiza,the CDS was 696 bp,encoding 231 amino acids.2.SmbHLH74 was mainly and highly expressed in root;MeJA can significantly inhibit the transcription of SmbHLH74.3.The contents of cryptotanshinone,tanshinone I and tanshinone IIA in SmbHLH74-OE4、SmbHLH74-OE12 and SmbHLH74-OE19 transgenic hairy roots were significantly reduced;The expression levels of most key enzyme genes in the tanshinone biosynthesis pathway were analyzed by RT-qPCR in SmbHLH74-OE4 transgenic hairy root,the expression of SmHMGR1,SmGGPPS1 and SmCYP76AH1 were substantialy and significantly reduced in transgenic hairy root when compared with the wild type.Thus these three genes were chosen as the candidate target genes of SmbHLH74.4.In order to reveal the molecular mechanism of the inhibition effect of SmbHLH74 transcription factor on tanshinones accumulation,ChIP-qPCR and EMSA experiments demonstrated that SmbHLH74 can directly bind to the promoters of SmHMGR1,SmGGPPS1 and SmCYP76AH1 both in vivo and in vitro;further dual luciferase reporter(D-luc)assay found that SmbHLH74 directly inhibited the activity of promoters of SmHMGR1,SmGGPPS1 and SmCYP76AH1.5.JA can significantly inhibit the transcription of SmbHLH74.SmMYC2,the key transcription factor of JA signaling,may be the upstream direct molecule of SmbHLH74,and this was verified by Y1 H and EMSA assays;GUS reporter assay found that SmMYC2 negatively regulated the transcription of SmbHLH74.Taken together,this study revealed that SmbHLH74 directly inhibited the transcription of SmHMGR1,SmGGPPS1 and Sm CYP76AH1,thereby negatively regulated the biosynthesis of tanshinones.JA inhibiting the transcription of SmbHLH74 was mediated directly by SmMYC2,thereby JA can release,at least partially,the inhibition effect of SmbHLH74 on tanshinones accumulation.