Cloning And Characterization Of Related Genes Involved In The Regulation Of Phenolic Acids Biosynthesis In Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge is one of the most widely used traditional herbal medicines forthe treatment of cardiovascular and cerebrovascular diseases in China. It is one of renascentherbs which belong to the Labiatae family and is mainly distributed in China and some otherAsian countries. The bioactive components of S. miltiorrhiza are divided into two groups, thewater-soluble phenolic acids and the lipid-soluble tanshinones. The water-soluble phenolicacids mainly contain caffeic acid, danshensu ((r)-a,3,4-trihydroxybenzenepropanoic acid),rosmarinic acid (RA) and salvianolic acid B (SAB). The lipid-soluble tanshinones includetanshinone I, tanshinone IIA, dihydrotanshinone I, cryptotanshinone and etc., which belong toa group of diterpenes with an abietane-type skeleton. In recent years, the water-solublephenolic acids have attracted attention for their marked pharmacological activities coupledwith their traditional use from herbs steeped in boiling water in China. In this study, sterileplantlets and hairy roots were used as materials to investigate the regulatory mechanism ofphenolic acids biosynthesis in S. miltiorrhiza. The main content was as follows:1. Four R2R3MYB genes were isolated by the homology-based cloning, which were termed as SmMYB39(GenBank accession No.: KC771280), SmMYB29(GenBank accession No.:KC213792), SmMYB87(GenBank accession No.:KC771280) and SmMYB36(GenBank accession No.:JX997333), respectively. Analysis of these genomic DNA showed that SmMYB39contained two exons and one intron, SmMYB29contained none intron, SmMYB87contained three exons and two introns, SmMYB26contained three exons and two introns. The four genes harboured open reading frames (ORFs) with length of693bp,321bp,732bp and960bp, and coded230amino acids,106amino acids,243amino acids and319amino acids, respectively. R2and R3repeats were found at N terminal of each amino acid sequence, implying that they were all R2R3MYB proteins.2. A1137bp sequence of SmMYB365’ flanking sequence was isolated with the Genome Walking method. The sequence was analysised by the PlantCARE software. The result showed that this sequence contained a TATA-box30bp upstream from the transcriptional start site, which is the common site of this motif in the eukaryotic gene promoters. It contained many CAAT-boxes and they dispersedly distribute in the whole sequence. Besides, there were several optical sensing elements in this sequence, such as ACE, AE-box, AT1-motif, Box4, Box I, G-Box, GT1-motif, I-box, MRE, as-2-box and chs-CMA2a, indicating that the exression of this gene may be affected by light.3. QRT-PCR was performed to determine expression levels of SmMYB39, SmMYB29, SmMYB87and SmMYB36in different tissues (root, stem, leaf and flower) of two-year-old flowering S. miltiorrhiza. Results showed that they expressed in all the tissues tested with different patterns. SmMYB39represented the highest expression in stem, followed by that in the leaf and flower, and showed the lowest expression in root. SmMYB29also represented the highest expression in stem, followed by that in the flower, and showed the lowest expression in root. Expression of SmMYB87in root was slight lower than that in other three organs, and transcript levels of this gene in stem, leaf and flower were similar. Expression of SmMYB36in root is the lowest among the four organs tested, and transcript levels of this gene in other three organs were almost identical.4. To examine the subcellular localization of target proteins, the open reading frames of their nucleotide sequences were fused to the5’-terminus of the GFP reporter gene under control of the CaMV35S promoter, respectively. The recombinant constructs of the SmMYB-GFP fusion gene and GFP alone were introduced into onion epidermal cells by particle bombardment, respectively. Results showed that SmMYB39and SmMYB29were located in nucleus, SmMYB87and SmMYB36were located both in nucleus and cytomembrane.5. Overexpression and RNAi vectors of the target genes were constructed and introduced into S. miltiorrhiza with the Agrobacterium tumefaciens mediated genetic transformation system. Compared with the untransformed plantlets and vector-control lines, accumulations of phenolic acids were dramatically (P <0.05) reduced in SmMYB39-overexpressing lines. While in the transgenic SmMYB39-RNAi lines, production of the target metabolites was markedly (P <0.05) up-regulated. Changes of metabolites content in transgenic lines implied that SmMYB39was involved in the RA biosynthetic pathway and acted as a repressor of phenolic acids production. Transcript levels and enzyme activies of key enzymes in the RA pathway were determinated. Two key enzyme genes, C4H and TAT, whose transcripts and enzyme activities were all regulated by SmMYB39were identified as the target genes of this transcription factor.6. Three conserved CHS fragments were isolated with degenerate PCR, which were termed as SmCHS1(GenBankaccession No.：KF255832), SmCHS2(GenBankaccession No.： KF255833) and SmCHS3(GenBankaccession No.：KF255834), respectively. RNAi vector was constructed and transgenic S. miltiorrhiza hairy roots were obtained through the Agrobacterium tumefaciens (ATCC15834) mediated genetic transformation system. Then salicylic acid (SA) was added to the wild type, empty control and CHS RNAi hairy root cultures to investigate the effects of CHS silenceing and/or elicitor treatment on the accumulation of phenolic acids. Results showed that both CHS silenceing and SA enhanced the production of phenolic acids in the hairy roots. And the content in the SA treated transgenic cultures was much higher than that in the CHS silenceing and SA treated wild type cultures. Accumulations of RA and SAB in the SA treated transgenic cultures were about3.89-fold and4.26-fold of that in the untreated wild type cultures.7. Methyl jasmonate and hyphal extracts from Sclerotium rolfsii Sacc were served as “enhancer” and “repressor”, respectively, to examine the relationship between accumulation of phenolic compounds and enzymes in the two parallel pathways of RA pathway in S. miltiorrhiza hairy root cultures. Responses of enzymes in the tyrosine-derived pathway, at both the gene transcript and enzyme activity levels, showed a better consistency with alterations of phenolic compounds content after the two elicitor treatments. Our study implied that compared with enzymes in the phenylpropanoid pathway, enzymes in the tyrosine-derived pathway are more correlated to RA and SAB biosynthesis in S. miltiorrhiza hairy roots.