The Effect Of SmMYC2 On The Synthesis Of Phenolic Acids And Root Biomass In Salvia Miltiorrhiza

MYC2 is a core transcription factor in the plant response to jasmonates.It also functions in secondary metabolism and various processes for growth and development.The roles of JA in controlling genes participated in the synthesis of secondary metabolites are well-established.MYC2 positively or negatively regulates secondary metabolism during JA signaling in a species-specific manner.For example,it can positively regulate the biosynthesis of flavonoid(e.g.,anthocyanin)but inhibit JA-responsive biosynthesis of Trp-derived indole-glucosinolates in Arabidopsis plants with JA treatment.In Nicotiana tabacum,NtMYC2a and NtMYC2b control multiple JA-inducible steps in nicotine biosynthesis.MdMYC2 positively regulates flavonoid biosynthesis by modulating the expression of positive regulators in Malus domestica during JA signaling.JA inhibits root growth in Arabidopsis.MYC2,as a core factor in the JA signaling pathway,positively regulates j asmonate-induced root growth inhibition.Salvia miltiorrhiza Bunge is a very useful medicinal plant and has been applied to treat various maladies,especially coronary and cerebrovascular diseases.S.miltiorrhiza is Labiatae perennial herb.As one of the best-known Chinese traditional herbs,its root has been clinically used for more than 2000 years.Active ingredients in S.miltiorrhiza are classified as lipophilic diterpene quinone pigments,generally known as tanshinones and hydrophilic phenolic acids,which are mainly rosmarinic acid(RA)and its derivative salvianolic acid B(Sal B).In this study,main active ingredient content analysis and transcriptome analysis of transgenic S.miltiorrhiza over-expressed MYC2 were performed.Besides,we did yeast one-hybrid analysis to study target genes on the SalB synthetic pathway directly regulated by SmMYC2.Finally,the morphology of SmMYC2 over-expressed Salvia miltiorrhiza was observed and we measured its biomass.Our study wants to explore the effect of SmMYC2 on the synthesis of phenolic acids and root biomass in Salvia miltiorrhiza.In this study,the main contents and results are listed as follows:1.We have already generated transgenic S.miltiorrhiza plants that over-expressed SmMYC2 and we wanted to screen for the higher expression lines.Real-time quantitative PCR demonstrated that the expression levels of SmMYC2 in OEM-3、OEM-8、OEM-12 and OEM-14 were 2.7-fold,3.7-fold,8.4-fold and 3.1-fold of that in the control line(CK),respectively.So we chose OEM-8 and OEM-12 for further analysis.2.HPLC was used to characterize the production of phenolic acids and tanshinones in plants over-expressed SmMYC2.Overexpression tended to improve their biosynthesis.In the roots of two-month-old transgenic tissue culture,RA and its dimer Sal B were the major components.The content of RA and Sal B in OEM-12 were 2.46-fold and 1.88-fold of those in CK.In OEM-8,concentrations of RA and Sal B were approximately 2.40-and 1.51-fold of those in CK.The two tanshinones investigated here are key active ingredients in danshen.However,neither was detected by HPLC in the two-month-old OEM lines and control plants3.The global expression profiles of OEM-12 versus CK was compared.Two cDNA libraries were sequenced by Illumina deep sequencing to obtain approximately 31.26 and 25.93 million high-quality clean reads for OEM-12 and CK,respectively.Each read averaged 296 bp long.We analyzed the expression of unigenes using Bowtie and RSEM software.Transcriptome analysis identified 2694 DEGs,with 1540 unigenes being up-regulated and 1154 down-regulated in OEM-12 when compared with expression in CK.Our KEGG analysis enabled us to identify 20 significantly enriched metabolic pathways for those DEGs,including biosynthetic pathways for phenolics,phenylalanine,tyrosine,and JA.To validate the RNA-seq data,we examined 12 genes involved in phenolic acid biosynthesis(TAT1,CYP98A14,HPPR1,C4H1,RAS1),the JA signaling pathway(JAZ1 and JAZ3),tanshinone biosynthesis(DXS3,GGPPS1 and HMGR4)and flavonoids biosynthesis(FLS and F3’5’H).Their expression patterns,as detected by qRT-PCR,were consistent with those obtained from the RNA-Seq data.Overall,this qRT-PCR analysis confirmed that the RNA-Seq results were statistically reliable and comparable to transcriptomic data.4.MYC2 binds to G-box in gene promoter region to regulate gene expression.We analyzed the promoter sequences of 28 putative genes for enzymes in the pathway for salvianolic acid biosynthesis and found that many,e.g.,SmTAT1,SmHPPR1,SmCYP98A14,and SmRAS2,contain G-box sequences in their promoters.This suggests that MYC2 directly binds to the promoter of each of those genes to regulate their expression.Based on our transcriptome results and quantitative results,the promoter regions of SmPAL1,SmTAT1 and SmCYP98A14 were constructed on the pHIS2 vector respectively,and SmMYC2 was constructed on the pGADT7-AD vector.We used yeast one-hybrid technique to verify whether the promoter regions of SmPAL1,SmTAT1 and SmCYP98A14 interact with SmMYC2.The results showed that the promoter regions of SmPAL1,SmTAT1 and SmCYP98A14 all interact with SmMYC2.5.Root phenotype of two-month-old transgenic lines overexpressed SmMYC2 and CK were observed.Compared to CK,root growth of OEM-12 strain was inhibited.Then the root biomass was measured.As a result,the biomass of CK was significantly higher than that of OEM-12.The average biomass of CK was 146.36±30.06 mg,while the average biomass of OEM-12 was 88.07±8.93 mg.In this study,the content of phenolic acids in SmMYC2 overexpressed transgenic Salvia miltiorrhiza strains was determined,and transcriptomic analysis,yeast one-hybrid experiment were performed.Our study preliminary explored the molecular mechanism by which SmMYC2 regulates phenolic acid biosynthesis in S.miltiorrhiza and it provides a good foundation for further understanding of the molecular mechanism regulated by SmMYC2.