Functional Characterization And Metabolic Regulatory Mechanism Analysis Of SmMYB36 In Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge is the medical model plant,and its dried root and rhizome are useful for preventing and treating various diseases,such as coronary heart disease,atherosclerosis and angina pectoris.Phenolic acids and tanshinones are two major bioactive components in S.miltiorrhiza.In recent five years,much more attention has been paid to the illustration of metabolic pathways and biosynthetic regulation of secondary metabolites using modern biotechnology in S.miltiorrhiza.In this research,we overexpressed SmMYB36 in hairy roots of S.miltiorrhiza,and aimed to explore the regulatory mechanism of SmMYB36 in the biosynthesis of active components.The main contents are as follows:1.SmMYB36 was cloned and identified as a gene coding an R2R3-MYB transcription factor by sequence alignments and motif analysis.The predicted orthologous genes of SmMYB36 were found by bidirectional best BLAST hits,and AtMYB23 was the orthologous gene of SmMYB36 in Arabidopsis thaliana.Phylogenetic analysis and motif analysis demonstrated that SmMYB36 was a novel member in evolution.2.The plasmids pA7-GFP-SmMYB36 and pA7-GFP were transiently transformed into onion epidermis using the gene gun method to determine the subcellular localization of SmMYB36.The GFP fluorescence of the control existed in the nucleus and cytoplasm.The GFP fluorescence of SmMYB36 was intensive in the nucleus and dispersive in the cytoplasm.3.The hairy roots were derived from S.miltiorrhiza sterile leaves infected by Agrobacterium rhizogenes strain ATCC15834 containing the plasmid pK7WG2R-SmMYB36.The control hairy roots were yellowish lines,and the SmMYB36-overexpressed hairy roots were brick-red lines.HPLC,GC and physiological assay indicated that the overexpression of SmMYB36 inhibited the accumulation of rosmarinic acid,salvianolic acid B,total phenolics,total flavonoids and oleic acid significantly(P<0.05)but promoted the accumulation of dihydrotanshinone I,cryptotanshinone,tanshinone I,tanshinone II A and linoleic acid markedly(P<0.05)in S.miltiorrhiza hairy roots.4.The expression levels of key genes of the phenolic acid biosynthesis pathway and tanshinone biosynthetic pathway in SmMYB36-overexpressed hairy roots were detected using quantitative RT-PCR.The transcript-level changes in metabolic pathway genes were in accordance with changes in metabolite content.Most candidate genes of the phenylpropanoid pathway(PAL1,C4H1 and 4CL2)and tyrosine pathway(TAT1 and HPPR1)were down-regulated markedly(P<0.05),and the transcript level of methylerythritol phosphate pathway genes(DXS1,DS2,DXR,MCT,MDS,HDS,CMK and HDR1)and downstream pathway genes(GGPPS1,CPS1,KSL1 and CYP76AHI)were enhanced substantially due to the overexpression of SmMYB36.5.The prokaryotic expression plasmid pET32a-SmMYB36 were constructed and expressed in E.coli BL21.The target proteins were obtained after purification by Ni-NTA Resin and identification by western blot assays.The promoter sequences were obtained based on the genome sequence of S.miltiorrhiza,and the MYB response elements(MBS1,MBS2,MBS3,MRE,MBS ? and MBS ?)of promoters were found by PlantCARE database.The element sequences of promoters were synthesized as probes.The Electrophoretic Mobility Shift Assay was conducted to explore whether SmMYB36 could interact with probes in vitro.SmMYB36 could interact with three probes(MBS1,MBS ? and MBS ?).