Identification Of MYB-bHLH-WD40 Ternary Complex Regulating Salvianolic Acid B Biosynthesis In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is an important medicinal plant.Its dry roots or rhizomes have good effects on removing blood stasis,cooling the blood,treating boils,activating blood to promote menstruation,etc.They have been widely used for prevention and treatment of the cardiovascular,cancer,and a variety of inflammatory diseases in clinical medication.The active ingredients of Salvia miltiorrhiza are divided into two groups:lipid-soluble tanshinones and water-soluble phenolic acids.Based on the traditional use of herbs by decocting with water,the main active ingredients of the S.miltiorrhiza are considered to be phenolic acids,which are related to the two metabolic pathways of tyrosine and phenylpropane.In recent years,a large number of enzyme genes and transcription factors involving in phenolic acid biosynthesis have been cloned,and their functions have been studied preliminarily.Previous studies have shown that SmTTG1(WD40)and SmMYC(bHLH51)regulated the biosynthesis of salvianolic acid B by affecting the expression of several key enzyme genes involved in phenolic acid synthesis pathway.It has been shown that the MYB,bHLH and WD40 transcription factors can form the MYB-bHLH-WD40 complex which could significantly affect the accumulation of secondary metabolites by binding to the promoters of structural genes of the secondary metabolism pathway in Arabidopsis and other model plants.However,the endogenous MBW transcription complex of S.miltiorrhiza involving in salvianolic acid B biosynthesis is unknown.In this study,the full length of SmTTGl gene of S.miltiorrhiza was cloned and the recombinant plasmid pCBKT7-SmTTGl was constructed to screen the cDNA library of S.miltiorrhiza.In the present study,the SmTTG 1-SmMYB 111-SmMYC(MBW)transcription complex,which may be involved in the biosynthesis of salvianolic acid B was identified for the first time.The results lay a foundation for elucidating the molecular mechanism of the phenolic acid biosynthesis in S.miltiorrhiza.The main results were as follows:1.The coding sequence of SmTTG1 was cloned and inserted into the expression vector pGBKT7-GW(BD)by gateway technology to construct the SmTTG 1-BD bait expression vector.A test of yeast OD600 value for toxicity showed that SmTTG1-BD fusion protein has no toxicity.In addition,a test for autoactivation also showed that SmTTGl-BD bait recombination plasmid has no autoactivation on SD/-Trp/-His/X-?-gal,SD/-Trp/-Ade/X-a-gal nutritional deficient medium plates.This showed that SmTTG1-BD bait recombination plasmid can be used for screening cDNA library of S.miltiorrhiza.2.The interactive genes were screened from cDNA library of S.miltiorrhiza by the bait vector of SmTTG1-BD and positive clones were sequenced and analyzed by BLAST alignment in the NCBI database.Using Uniprot online web site,49 positive clones were annotated from biological processes,cellular components and molecular functions.The results showed that the 49 positive clones included MYB transcription factor(PPI7),5 genes related to abiotic stress,1 cinnamic acid-4-hydroxylase(C4H)involved in the regulation of phenolic acid biosynthesis,4 genes which involved in photosynthesis and other catalytically active enzyme genes.3.A 795 bp unigene was obtained by BLAST alignment in transcriptome database of S.miltiorrhiza with PPI7 gene sequence,and the full-length DNA sequence and cDNA sequence were cloned using PCR methods.The total length of PPI7 was 973 bp,which contained an intron and an open reading frame(ORF)of 795 bp encoding 264 amino acids.The amino acids encoded by the gene shared high similarity with the AtMYB111 of Arabidopsis thaliana and belonged to the R2R3-MYB subfamily member.We named it SmMYB111 Real-time PCR results showed that SmMYBlll was expressed in roots,stems,leaves and flowers of S.miltiorrhiza,and the expression level was the highest in roots.PlantCARE datebase analysis showed that the 2037 bp-long 5,flanking sequence of SmMYB111 included multiple hormone responsive elements and MYB binding sites.The quantitative real-time PCR showed that the expression of SmTTG1 could be induced by MeJA,SA,GA and light.4.To research the subcellular localization of the target proteins,SmMYB111,SmTTG1,and SMMYC,their coding DNA sequences(CDSs)without stop codons were cloned into pEarleyGate103 vector by Gateway technology,respectively.Then the recombinant plasmids were introduced into onion epidermal cells by particle bombardment using a helium-driven particle accelerator.Results showed that SmMYC and SmMYB111 were located in nucleus,and SmTTG1 was located both in nucleus and cytomembrane.5.The coding DNA sequences(CDSs)of SmMYB111,SmTTG1,and SmMYC were cloned into yeast expression vector and BIFC expression vector by Gateway technology,respectively.Yeast two-hybrid assay and bimolecular fluorescence complementary assay showed that SmMYB1 11 protein may interact with SmMYC and SmTTGl,but SmTTG1 did not interact with SmMYC.The SmMYB1 11 may interact with SmTTGl or SmMYC to form SmMYC-SmMYB1 11-SmTTGl ternary complex which probably regulated the biosynthesis of salvianolic acid B.In this study,we used the SmTTG1 as a bait protein to screen the cDNA library of S.miltiorrhiza and obtained a R2R3-MYB transcription factor.Further,we identified the endogenous SmMYC-SmMYB111-SmTTGl protein complex probably related to the synthesis of salvianolic acid B,which laid a foundation for clarifying the molecular mechanism of phenolic compounds biosynthesis in S.miltiorrhiza,and provided new ideas and strategies for molecular breeding of S.miltiorrhiza.