methyl-jasmonate and tanshinone

The present study characterizes the 5' regulatory region of the

Topics: Abietanes; Acetates; Cyclopentanes; Oxylipins; Salvia miltiorrhiza

Topics: Abietanes; Acetates; Cyclopentanes; F-Box Proteins; Gene Expression Regulation, Plant; Hydroxybenzoates; Kelch Repeat; Oxylipins; Phylogeny; Plant Roots; Salvia miltiorrhiza; Transcription Factors

MIR396b had been cloned and overexpressed in Salvia miltiorrhiza hairy roots. MiR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to regulate cell growth and secondary metabolism in S. miltiorrhiza hairy roots. Danshen (Salvia miltiorrhiza Bunge) is a valuable medicinal herb with two kinds of clinically used natural products, salvianolic acids and tanshinones. miR396 is a conserved microRNA and plays extensive roles in plants. However, it is still unclear how miR396 works in S. miltiorrhiza. In this study, an smi-MIR396b has been cloned from S. miltiorrhiza. Overexpression of miR396b in danshen hairy roots inhibited hairy root growth, reduced salvianolic acid concentration, but enhanced tanshinone accumulation, resulting in the biomass and total salvianolic acids respectively reduced to 55.5 and 72.1% of the control and total tanshinones increased up to 1.91-fold of the control. Applied degradome sequencing, 5'RLM-RACE, and qRT-PCR, 13 targets for miR396b were identified including seven conserved SmGRF1-7 and six novel ones. Comparative transcriptomics and microRNomics analysis together with qRT-PCR results confirmed that miR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to mediate the phytohormone, especially gibberellin signaling pathways and consequentially resulted in the phenotype variation of miR396b-OE hairy roots. Furthermore, miR396b could be activated by methyl jasmonate, abscisic acid, gibberellin, salt, and drought stresses. The findings in this study indicated that smi-miR396b acts as an upstream and central regulator in cell growth and the biosynthesis of tanshinones and salvianolic acids, shedding light on the coordinated regulation of plant growth and biosynthesis of active ingredients in S. miltiorrhiza.

Topics: Abietanes; Abscisic Acid; Acetates; Alkenes; Anthocyanins; Binding Sites; Biomass; Cell Proliferation; Cyclopentanes; Droughts; Gene Expression Profiling; Gene Expression Regulation, Plant; Gene Ontology; Gene Regulatory Networks; Gibberellins; MicroRNAs; Oxylipins; Phylogeny; Plant Proteins; Plant Roots; Plants, Genetically Modified; Polyphenols; Propanols; RNA Stability; Salt Stress; Salvia miltiorrhiza; Secondary Metabolism; Terpenes; Transcription Factors; Transcription, Genetic; Transcriptome

The purpose of this study is to reveal the impact of the plant hormone salicylic acid (SA) and methyl jasmonate (MeJA) on the growth, effective components accumulation, and related gene expression of the hairy root of Salvia przewalskii Maxim. Various concentrations of SA (0, 25, 50, 100, 200 μM) or MeJA (0, 50, 100, 200, 400, 600 μM) were added to the culture medium of Salvia przewalskii Maxim. Low concentrations of SA promoted the growth of hairy root, while a high concentration inhibited it. 0 to 400 μM MeJA promoted the growth of hairy root, but 600 μM MeJA starts to inhibit its growth. 50 μM SA and 400 μM MeJA significantly enhanced the production of caffeic acid, rosmarinic acid, salvianolic acid B, cryptotanshinone, and tanshinone IIA. In general, 50 μM SA can be used to accumulate of tanshinone in hairy roots of S. przewalskii with 6 days. 400 μM MeJA can be used to accumulate of phenolic acids in hairy roots of S. przewalskii with 3 days. The selected genes in the tanshinone and phenolic acid biosynthetic pathway were upregulated with elicitation. To obtain a higher yield and content of secondary metabolites, it is advisable to use 50 μM SA or 400 μM MeJA as the optimal doses to cultivate the hairy root of S. przewalskii. This study provides, for the first time, an efficient tanshinone and phenolic acid production method for S. przewalskii.

Topics: Abietanes; Acetates; Benzofurans; Caffeic Acids; Cinnamates; Cyclopentanes; Depsides; Dose-Response Relationship, Drug; Gene Expression Regulation, Plant; Hydroxybenzoates; Oxylipins; Phenanthrenes; Plant Growth Regulators; Plant Proteins; Plant Roots; Rosmarinic Acid; Salicylic Acid; Salvia; Time Factors

Tanshinones are important bioactive components in Salvia miltiorrhiza and mainly accumulate in the periderms of mature roots. Tanshinone biosynthesis is a complicated process, and little is known about the third stage of the pathway. To investigate potential genes that are responsible for tanshinone biosynthesis, we conducted transcriptome profiling analysis of two S. miltiorrhiza cultivars. Differential expression analysis provided 2,149 differentially expressed genes (DEGs) for further analysis. GO and KEGG analysis showed that the DEGs were mainly associated with the biosynthesis of secondary metabolites. Weighted gene coexpression network analysis (WGCNA) was further performed to identify a "cyan" module associated with tanshinone biosynthesis. In this module, 25 cytochromes P450 (CYPs), three 2-oxoglutarate-dependent dioxygenases (2OGDs), one short-chain alcohol dehydrogenases (SDRs) and eight transcription factors were found to be likely involved in tanshinone biosynthesis. Among these CYPs, 14 CYPs have been reported previously, and 11 CYPs were identified in this study. Expression analysis showed that four newly identified CYPs were upregulated upon application of MeJA, suggesting their possible roles in tanshinone biosynthesis. Overall, this study not only identified candidate genes involved in tanshinone biosynthesis but also provided a basis for characterization of genes involved in important active ingredients of other traditional Chinese medicinal plants.

Topics: Abietanes; Acetates; Biosynthetic Pathways; Cyclopentanes; Cytochrome P-450 Enzyme System; Dioxygenases; Gene Expression Regulation, Plant; Medicine, Chinese Traditional; Oxylipins; Plant Proteins; Plant Roots; Plants, Medicinal; RNA-Seq; RNA, Plant; Salvia miltiorrhiza; Transcription Factors

Phenolic acids are important secondary metabolites produced in the Chinese medicinal plant Salvia miltiorrhiza, but little is known about the transcription factors involved in the regulation of tanshinone and phenolic acid biosynthesis. Here, a novel AP2/ERF transcription factor SmERF115 was isolated and functionally characterized. SmERF115 was most responsive to methyl jasmonate (MeJA) treatment and was localized in the nucleus. The phenolic acid production was increased in SmERF115-overexpressing hairy roots, but with a decrease in tanshinone content. In contrast, silencing of SmERF115 reduced the phenolic acid level, but increased tanshinone content. The expression of the key biosynthetic gene SmRAS1 was up-regulated in SmERF115 overexpression lines but was down-regulated in SmERF115-RNAi lines. Yeast one-hybrid (Y1H) assay and EMSA showed that SmERF115 directly binds to the promoter of SmRAS1, while dual-luciferase assays showed that SmERF115 could activate expression of SmRAS1 in vivo. Furthermore, global transcriptomic analysis by RNA sequencing revealed that expression of other genes such as PAL3, 4CL5, TAT3, and RAS4 was also increased in the overexpression line, implying that they were potentially involved in the SmERF115-mediated pathway. Our data show that SmERF115 is a positive regulator of phenolic acid biosynthesis, and may be a potential target for further metabolic engineering of phenolic acid biosynthesis in S. miltiorrhiza.

Topics: Abietanes; Acetates; Cyclopentanes; Gene Expression Regulation, Plant; Hydroxybenzoates; Oxylipins; Plant Proteins; Salvia miltiorrhiza; Transcription Factors

Salvia castanea Diels f. tomentosa Stib. is an endemic medicinal plant distributed in China, and the notably high content of tanshinone IIA in the root is proven effective for the therapy of heart diseases. Hairy root induction of this Salvia species was inoculated with Agrobacterium rhizogenes strain ATCC 15834. Transformed hairy root was cultured in 6,7-V liquid medium for growth kinetics assessment and elicitation. An S curve was present in the hairy root cultures based on the fresh and dry weights with an interval of 3 days. An optimum concentration of the applied elicitors (15 μM Ag(+), 200 μM methyl jasmonate, and 200 mg l(-1) yeast extract elicitor) benefitted both the growth status and tanshinone accumulation in the hairy root cultures. Tanshinone IIA contents were mostly stimulated 1.8-fold and 1.99-fold compared with the control by Ag(+) and methyl jasmonate elicitation, respectively. Yeast extract dramatically enhanced dry mass accumulation, while it promoted cryptotanshinone content of 2.84 ± 0.33 mg g(-1) dry weight at most in the hairy root cultures. Selected elicitors diversely influenced tanshinone accumulation in the time courses of hairy root cultures within 7 days. Furthermore, transcripts of selected genes in the tanshinone biosynthetic pathway were remarkably upregulated with elicitation. Yeast extract elicitor heightened 13.9-fold of isopentenyl diphosphate isomerase expression level at 12 h, while it increased 16.7-fold of geranylgeranyl diphosphate synthase transcript at 24 h compared with that of the control, which was more effective than Ag(+) and methyl jasmonate. This study provided a convenient hairy root culture system of S. castanea Diels f. tomentosa Stib. for tanshinone production for the first time.

Topics: Abietanes; Acetates; Biomass; Biosynthetic Pathways; Cyclopentanes; Gene Expression Regulation, Plant; Genes, Plant; Kinetics; Oxylipins; Plant Roots; Saccharomyces cerevisiae; Salvia; Silver; Time Factors; Tissue Culture Techniques

Jasmonic acid (JA) is an important plant hormone involved in regulation of many aspects of plant growth and development including secondary metabolism and JASMONATE ZIM-DOMAIN (JAZ) proteins are key components in JA signal processes. In this study, two new JAZ genes named SmJAZ3 and SmJAZ9 were cloned from S. miltiorrhiza hairy roots and characterized. Expression profiles under methyl jasmonate (MJ) treatment revealed that SmJAZ3 and SmJAZ9 were both MJ-responsive. Subcellular localization assay showed that SmJAZ3 was located in nucleus while SmJAZ9 was preferentially in nucleus. Expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots differently affected the production of tanshinone. Over-expression of SmJAZ3 or SmJAZ9 in hairy roots produced lower level of tanshinone compared with the control, tanshinone production was as low as 0.077 mg/g DW in line SmJAZ3-3 and 0.266 mg/g DW in line SmJAZ9-22. Whereas, down-regulation of SmJAZs enhanced tanshione production, the content of tanshinone increased to 2.48 fold in anti-SmJAZ3-3 line, and 1.35-fold in anti-SmJAZ9-23 line. Our work indicated that SmJAZ3 and SmJAZ9 are involved in regulation of tanshinone biosynthesis and act as repressive transcriptional regulators in the JA signaling pathway, which paves the way to further dissect molecular mechanism in details in the future.

Topics: Abietanes; Acetates; Arabidopsis; Arabidopsis Proteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cloning, Molecular; Cyclopentanes; Gene Expression Regulation, Plant; Oxylipins; Phylogeny; Plant Growth Regulators; Plant Roots; Repressor Proteins; Salvia miltiorrhiza; Sequence Analysis, DNA

Tanshinones are major bioactive diterpenoids of Salvia miltiorrhiza roots used for the treatment of cardiocerebral diseases. To develop effective elicitation and bioprocess strategies for the enhanced production of tanshinones, ultraviolet-B (UV-B) irradiation and methyl jasmonate (MeJA) elicitation were applied alone or in combination respectively in S. miltiorrhiza hairy root cultures. Our results showed 40-min UV-B irradiation at 40μW/cm(2) stimulated tanshinone production without any suppression of root growth, suggesting a new effective elicitor to S. miltiorrhiza hairy root cultures for tanshinone production. Moreover, the combined treatment of UV-B irradiation and MeJA exhibited synergistic effects on the expression levels of 3-hydroxy-3-methylglutaryl-CoA reductase (SmHMGR) and geranylgeranyl diphosphate synthase (SmGGPPS) genes in the tanshinone biosynthetic pathway. When hairy roots of 18-day-old cultures were exposed to the combined elicitation for 9days, the maximum production of tanshinone reached to 28.21mg/L, a 4.9-fold increase over the control. The combined elicitation of UV-B and MeJA was firstly used to stimulate the production of biologically important secondary metabolites in hairy root cultures.

Topics: Abietanes; Acetates; Cyclopentanes; Oxylipins; Plant Roots; Salvia miltiorrhiza; Ultraviolet Rays

Salvia miltiorrhiza Bunge (Labiatae) is an emerging model plant for traditional medicine, and tanshinones are among the pharmacologically active constituents of this plant. Although extensive chemical and pharmaceutical studies of these compounds have been performed, studies on the basic helix-loop-helix (bHLH) transcription factors that regulate tanshinone biosynthesis are limited. In our study, 127 bHLH transcription factor genes were identified in the genome of S. miltiorrhiza, and phylogenetic analysis indicated that these SmbHLHs could be classified into 25 subfamilies. A total of 19 sequencing libraries were constructed for expression pattern analyses using RNA-Seq. Based on gene-specific expression patterns and up-regulated expression patterns in response to MeJA treatment, 7 bHLH genes were revealed as potentially involved in the regulation of tanshinone biosynthesis. Among them, the gene expression of SmbHLH37, SmbHLH74 and SmbHLH92 perfectly matches the accumulation pattern of tanshinone biosynthesis in S. miltiorrhiza. Our results provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factors in S. miltiorrhiza.

Topics: Abietanes; Acetates; Basic Helix-Loop-Helix Transcription Factors; Cyclopentanes; Genes, Plant; Oxylipins; Phylogeny; Plant Proteins; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; RNA, Plant; Salvia miltiorrhiza; Sequence Analysis, RNA; Transcriptome

The enzyme 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) is a terminal-acting enzyme in the plastid MEP pathway, which produce isoprenoid precursors. The full-length cDNA of HDR, designated SmHDR1 (Genbank Accession No. JX516088), was isolated for the first time from Salvia miltiorrhiza Bge. f. alba. SmHDR1 contains a 1389-bp open reading frame encoding 463 amino acids. The deduced SmHDR1 protein, which shows high identity to HDRs of other plant species, is predicted to possess a chloroplast transit peptide at the N-terminus and four conserved cysteine residues. Transcription pattern analysis revealed that SmHDR1 has high levels of transcription in leaves and low levels of transcription in roots and stems. The expression of SmHDR1 was induced by 0.1 mM methyl-jasmonate (MeJA) and salicylic acid (SA), but not by 0.1 mM abscisic acid (ABA), in the hairy roots of S. miltiorrhiza Bge. f. alba. Complementation of SmHDR1 in the Escherichia coli HDR mutant MG1655 ara < > ispH demonstrated the function of this enzyme. A functional color assay in E. coli showed that SmHDR1 accelerates the biosynthesis of β-carotene, indicating that SmHDR1 encodes a functional protein. Overexpression of SmHDR1 enhanced the production of tanshinones in cultured hairy roots of S. miltiorrhiza Bge. f. alba. These results indicate that SmHDR1 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza Bge. f. alba.

Topics: Abietanes; Abscisic Acid; Acetates; Amino Acid Sequence; beta Carotene; Chloroplasts; Cloning, Molecular; Cyclopentanes; DNA, Complementary; Escherichia coli; Gene Expression; Genes, Plant; Molecular Sequence Data; Mutation; Open Reading Frames; Oxidoreductases; Oxylipins; Plant Growth Regulators; Plant Proteins; Plant Structures; Salicylic Acid; Salvia miltiorrhiza

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O(2)(-) and H(2)O(2) were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.

Topics: Abietanes; Acetates; Cyclopentanes; Nitric Oxide; Nitroprusside; Oxylipins; Plant Roots; Reactive Oxygen Species; Salvia miltiorrhiza

Tanshinones, a group of active ingredients in Salvia miltiorrhiza, are derived from at least two biosynthetic pathways, which are the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the plastids. Abscisic acid (ABA) and methyl jasmonate (MJ) are two well-known plant hormones induced by water stress. In this study, effects of polyethylene glycol (PEG), ABA and MJ on tanshinone production in S. miltiorrhiza hairy roots were investigated, and the role of MJ in PEG- and ABA-induced tanshinone production was further elucidated. The results showed that tanshinone production was significantly enhanced by treatments with PEG, ABA and MJ. The mRNA levels of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS), as well as the enzyme activities of HMGR and DXS were stimulated by all three treatments. PEG and ABA triggered MJ accumulation. Effects of PEG and ABA on tanshinone production were completely abolished by the ABA biosynthesis inhibitor [tungstate (TUN)] and the MJ biosynthesis inhibitor [ibuprofen (IBU)], while effects of MJ were almost unaffected by TUN. In addition, MJ-induced tanshinone production was completely abolished by the MEP pathway inhibitor [fosmidomycin (FOS)], but was just partially arrested by the MVA pathway inhibitor [mevinolin (MEV)]. In conclusion, a signal transduction model was proposed that exogenous applications of PEG and ABA triggered endogenous MJ accumulation by activating ABA signaling pathway to stimulate tanshinone production, while exogenous MJ could directly induce tanshinone production mainly via the MEP pathway in S. miltiorrhiza hairy roots.

Topics: Abietanes; Abscisic Acid; Acetates; Biosynthetic Pathways; Cyclopentanes; Erythritol; Oxylipins; Plant Growth Regulators; Plant Roots; Plants, Genetically Modified; Polyethylene Glycols; Salvia miltiorrhiza; Sugar Phosphates

This study provides a desirable candidate gene resource (SmAOC) to increase the content of valuable natural products via appropriate JA pathway genetic engineering. Jasmonates (JAs) are important signal molecules in plants. They regulate transcripts of defense and secondary biosynthetic metabolite genes in response to environmental stresses. Currently, JAs are widely used as elicitors to improve the content of useful secondary metabolism in plants. Synthesis of the naturally occurring enantiomer of various jasmonates is catalyzed by allene oxide cyclase (AOC, EC 5.3.99.6). Here, we cloned and characterized the AOC gene (SmAOC) from Salvia miltiorrhiza. As expected, SmAOC expression was induced by abiotic stimuli such as methyl jasmonate (MeJA), ultraviolet radiation (UV) and low temperature (4 °C) in S. miltiorrhiza plantlets. To demonstrate whether the engineered internal JAs pool by overexpressing AOC gene could promote secondary metabolism production, the SmAOC was incorporated into S. miltiorrhiza hairy roots. The results revealed that SmAOC overexpression significant enhanced the yields of tanshinone IIA, rosmarinic acid (RA) and lithospermic acid B (LAB) in S. miltiorrhiza hairy roots. In addition, expression levels for key genes involved in the biosynthetic pathway of diterpenes and phenolic acids were also altered. These suggest that genetic manipulation of AOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis of JAs.

Topics: Abietanes; Acetates; Benzofurans; Cinnamates; Cloning, Molecular; Cold Temperature; Cyclopentanes; Depsides; Diterpenes; Escherichia coli; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Genetic Engineering; Genetic Vectors; Hydroxybenzoates; Intramolecular Oxidoreductases; Oxylipins; Plant Roots; Rosmarinic Acid; Salvia miltiorrhiza; Transgenes; Ultraviolet Rays

To observe the effects of a biotic elicitor fungal hyphae extract, an abiotic elicitor methyl jasmonate and their synergistic action on the accumulation of phenolic acids and tanshinones in Salvia miltiorrhiza hairy root.. Different elicitors were added to S. miltiorrhiza hairy root, which was subcultured for 21 days, the dry weight and contents of phenolic acids and tanshinones were determined at different harvest-time.. S. miltiorrhiza hairy root growth was significantly inhibited by all three treatments and the accumulation of cryptotanshinone and dihydrotanshinone were promoted by each elicition. As for the accumulation of phenolic acids, there were differences between fungal elicitor and methyl jasmonate treatments, they were promoted by methyl jasmonate while inhibited in a certain extent by fungal hyphae extract.. Fungal elicitor, methyl jasmonate and their synergistic action have significant influence on accumulation of components in S. miltiorrhiza hairy root, and the effect varies between phenolic acids and tanshinones. There is no correlation between production of water-soluble ingredients and fat-soluble components on the whole under three different treatments.

Topics: Abietanes; Acetates; Cyclopentanes; Fungal Proteins; Hydroxybenzoates; Membrane Glycoproteins; Oxylipins; Phytophthora; Plant Extracts; Plant Roots; Salvia miltiorrhiza

This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co2+, Ag+, Cd2+), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g(-1) versus 0.2 mg g(-1) in control). The stimulating effect was concentration-dependent, most significant at 25 microM of Ag and Cd and 100 mg l(-1) (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1-5.5 g l(-1) versus 8.9 g l(-1) in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.

Topics: Abietanes; Acetates; Biomass; Cell Culture Techniques; Cells, Cultured; Culture Media; Cyclopentanes; Hydrogen-Ion Concentration; Metals, Heavy; Oxylipins; Phenanthrenes; Salicylic Acid; Salvia miltiorrhiza

This paper firstly introduced the acquired full length cDNA of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from hairy roots of Salvia miltiorrhiza (Abbr: SmCMK, GenBank number: EF534309). Results of KEGG analysis showed that SmCMK was belong to the upstream of nonemevalonate pathway, the only one kinase of the pathway. The full-length cDNA was deduced as encoding 4-(cytidine 5'-diphospho)-2-C-methylerythritol kinase (designated as SmCMK), and the sequence had a 1493 bp including 5' UTR 71 bp and 3' UTR 232 bp, an open reading frame (ORF) encoding a protein of 396 amino acid residues. The deduced protein had isoelectric point (pI) of 6.78 and a calculated molecular weight about 43 kDa, similar to cloned diterpene of CMK from other species of plants such as Mentha piperita and Lycopersicon esculentum reported previously. Real time PCR results indicated that elicitors of MJ stimulated the increase of mRNA expression of SmCMK. At the same time, results of high performance liquid chromatography (HPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy root of Salvia miltiorrhiza were increased dramatically after treated with methyl jasmonate (MJ). This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by MJ. It proved primarily that the increased expression level of mRNA of SmCMK helps to enhance tanshinones' accumulation, which will be the basis for further study on the mechanism of gene regulation of secondary metabolism of tanshinones.

Topics: Abietanes; Acetates; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Conserved Sequence; Cyclopentanes; DNA, Complementary; Gene Expression Regulation, Plant; Genes, Plant; Molecular Sequence Data; Open Reading Frames; Oxylipins; Phenanthrenes; Phosphotransferases (Alcohol Group Acceptor); Plant Proteins; Plant Roots; Plants, Medicinal; RNA, Messenger; Salvia miltiorrhiza; Sequence Homology, Amino Acid

To study the effects of methyl jasmonate (MJ) on the accumulation and release of tanshinones in suspension cultures of Salvia miltiorrhiza hairy roots.. After 18 day's suspension culture of S. miltiorrhiza hairy roots induced by Agrobacterium rhizogenes ATCC15834, the chemical elicitor--methyl jasmonat was added into 6-7V suspension cultures and at the same time, tanshinones contents (including cryptotanshinone and tanshinone II(A)) on the day 2, 6 and 9, after dealing with MJ, was quantified by HPLC.. After dealing with MJ on the day 2, 6 and 9, the concentration of cryptotanshinone reached to 0.039, 0.204, 0.571 mg x g(-1) respectively,and tanshinone II(A) reached 0.251, 0.601 and 1.563 mg x g(-1) respectively. After 9 day's treatment by MJ, the maximum increase of cryptotanshinone and tanshinone II(A) were 23.8 fold and 6.2 fold higher than that of the control respectively.. MJ could stimulate the accumulation of tanshinones in S. miltiorrhiza and have released them into the culture medium.

Topics: Abietanes; Acetates; Culture Techniques; Cyclopentanes; Oxylipins; Phenanthrenes; Plant Growth Regulators; Plant Roots; Plants, Medicinal; Salvia miltiorrhiza