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When plants are subjected to mechanical wounding(MW)caused by insect feeding, extreme weather, and human factors, they rapidly initiate a series of response mechanisms at the transcriptional and metabolic levels, leading to changes in the content of phytohormone and secondary metabolites in plants. In this study, using the medicinal model plant Danshen(Salvia miltiorrhiza) as an example, the effect of MW on the metabolism of medicinal plants was evaluated. By virtue of qRT-PCR and LC-MS, the changes in the biosynthetic genes and contents of jasmonates(JAs) and tanshinones in response to leaf damage stimulation were detected to reveal the related patterns of transcription and metabolism in leaves and roots at different time points after MW treatment, thus exploring the response mechanism of Danshen to MW stress. The results showed that MW induction could transiently increase the expression of biosynthetic genes of Jas, with AOC and JAR beginning to increase and peaking at 2 h after induction, while AOS and OPR3 peaked at 4 h. Correspondingly, the content of OPDA, JA, and JA-Ile all peaked at 2 h. In the biosynthesis of tanshinones, the diterpene synthase genes CPS1 and KSL1 both peaked at 2 h, while the subsequent modification genes CYP450s all peaked at 4 h. The content of the four tanshinones showed a continuous increase trend within 8 h. This study provides a reference for revealing the research on secondary metabolite accumulation under MW stress and lays a foundation for further understanding the role of Jas in enhancing plant resistance, promoting the accumulation of active ingredients, and improving the quality of medicinal materials under MW stress.

The plant root-associated microbiomes include root microbiome and rhizosphere microbiome, which are closely related to plant life activities. Nearly 30% of photosynthesis products of plants are used to synthesize root compounds, there is evidence that root compounds regulate and significantly affect the root microbiome Tanshinones are the main hydrophobic components in Salvia miltiorrhiza. In order to study whether these compounds can regulate the root-associated microbiomes of S. miltiorrhiza, our study first identified a white root S. miltiorrhiza(BG) which contains little tanshinones. Retain of the fifth intron of tanshinones synthesis key enzyme gene SmCPS1 leading to the early termination of the SmCPS1 gene, and a stable white root phenotype. Further, wild type(WT) and BG were planted in greenhouse with nutrient soil(Pindstrup, Denmark) and Shandong soil(collected from the S. miltiorrhiza base in Weifang, Shandong), then high-throughput sequencing was used to analyze the root-associated microbiomes. The results showed that the tanshinones significantly affected the root-associated microbiomes of S. miltiorrhiza, and the impact on root microbiomes was more significant. There are significant differences between WT and BG root microbiomes in species richness, dominant strains and co-occurrence network. Tanshinones have a certain repelling effect on Bacilli which belongs to Gram-positive, while specifically attract some Gram-negative bacteria such as Betaproteobacteria and some specific genus of Alphaproteobacteria. This study determined the important role of tanshinones in regulating the structure of root-associated microbiomes from multiple angles, and shed a light for further improving the quality and yield of S. miltiorrhiza through microenvironment regulation.

Based on the theory of Q-marker, the hairy root of Salvia miltiorrhiza and S. miltiorrhiza in many provinces were studied. The relative expressions of SmCPS, SmKSL and CYP76AH1 genes in hairy roots were detected by real-time fluorescence quantitative PCR and the contents of tanshinoneⅡ_A, cryptotanshinone, tanshinoneⅠ, 1,2-dihydrotanshinone, ferruginol and miltiradiene were detected by UPLC and GC-MS, respectively. Statistical analysis shows as fllows: in the hairy root of S. miltiorrhiza, the content of miltiradiene and ferruginol is positively correlated with the content of tanshinone compounds in the downstream, and the relative expression of important genes in the biosynthetic pathway of tanshinone can reflect the content of tanshinone compounds to a certain extent; in many provinces of S. miltiorrhiza, the content of ferruginol and tanshinone compounds can also be found that there is a positive correlation between the contents. Based on the biosynthetic pathway of tanshinone compounds, which is a special index component in S. miltiorrhiza, this study focused on the important relationship between the upstream gene, the middle intermediate compound and the downstream tanshinone compound content of the biosynthetic pathway, and explored the possible research ideas of improving the quality marker system of S. miltiorrhiza, and then provided the possible research ideas for understanding and studying the quality marker of traditional Chinese medicine from the biosynthetic pathway.

Tanshinones are abietane-type norditerpenoid quinones that make up the main bioactive ingredients of traditional Chinese medicine Salvia miltiorrhiza. Cytochrome CYP450 plays an important role in the post-structural modification of tanshinone biosynthesis pathway. Long non-coding RNA( lncRNA) have been defined as transcripts longer than 200 nucleotides,which have been functionally characterized in regulating the growth and development,secondary metabolism and stress of medicinal plants. In this study,we perform a comprehensive identification of lncRNAs in response to tanshinone metabolism induced by yeast extract( YE) and Ag~+ S. miltiorrhiza hairy roots. Deep RNA sequencing was used to identify a set of different 8 942 lncRNAs,of which 6 755 were intergenic lncRNAs. We predicted a total of 1 115 814 lncRNA-coding gene pairs,including 122 lncRNA-coding gene as cis pairs. The correlation analysis between lncRNA and CYP450 related to tanshinone biosynthesis was carried out and a total of 16 249 lncRNA-CYP450 target gene pairs were identified. Further analysis with functional known CYP76 AH1,CYP76 AH3 and CYP76 AK1 involved in tanshinone biosynthesis,we also identified a set of 216 target genes. These candidate genes will be the important target in the downstream regulation mechanism analysis of the tanshinone biosynthesis pathway.

Rhizosphere bacteria play a vital role in plant nutrition absorption,growth and disease resistance. In this study,high-throughput sequencing technology was used to analyze the rhizosphere bacterial communities of Salvia miltiorrhiza and S. miltiorrhiza f. alba. Moreover,the function of dominant rhizosphere bacterial communities was analyzed. We found that Sphingobacteriales,Sphingomonadales and Nitrosomonadaceae were both dominant and specific bacteria in the rhizosphere of S. miltiorrhiza. The main functions of dominant rhizosphere bacteria communities in both species include promoting transformation of soil nutrients,improving plant immunity and ability of stress tolerance. This study was the first to compare rhizobacterial communities structure and function of S. miltiorrhiza and S. miltiorrhiza f. alba,which provided a new theoretical reference for studing the rhizosphere mechanism of healthy S. miltiorrhiza planting in the future.

Salviae Miltiorrhizae Radix et Rhizoma (Danshen) is one of the commonly used bulk medicinal materials in China. It is widely used in clinical practice, and has many pharmacological effects, such as antithrombotic，antibacterial，anti-inflammatory，anti-oxidative and immunomodulatory activities. The quality of Danshen determines the quality of the curative effect. The current wild resources of Danshen gradually decrease, the cultivation area is widely distributed, among them, Shandong, Sichuan, Anhui, Shanxi, Hebei, Henan and other provinces have large acreage for Danshen. However, germplasm, origin, cultivation, harvest, process and other factors have a certain impact on the active ingredients of Danshen, which lead to the quality of Danshen good and bad mixed. This paper is based on the systematic analysis of literature on quality evaluation of Danshen. To provide a new idea for the further research on the quality of Danshen, we summarized the main factors affecting the quality of Salvia miltiorrhiza that of germplasm, origin, cultivation, harvest, process, storage, product specifications and so on., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Danshen has a long history in China as a commen traditional Chinese medicine, but many other members of the Salvia family is also used as Danshen in particular regions. The name of Danshen were analyzed according to the color, efficacity and etc. To investigate the real origin of Danshen, the stalk, leaf, flower and radix in chronicle and pictures of ancient literature were deciphered. It showed that in the past dynasties, Salvia miltiorrhiza was the most likely plant used as Danshen. The rigin and quality evaluation of Danshen were also summerized, provided new insight and proof for the development and utilization of Danshen., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Basically, Dao-di hers are produced in specific area which has a long history, good quality, good medicine, curative effect. However genuine medicinal material area in history is not static, this makes the establishment of genuine medicinal material origin and the in-depth research be very difficult. This paper has profoundly analyzed the origin of different historical periods taking Astragalus membranaceus and Salvia miltiorrhiza as examples, and then summarized the reasons of herbal origin changes from the humanities, social and natural three aspects. This paper provides a basis for establishment and the further research of high-quality genuine producing area., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Plant cytochrome P450s (CYPs) are well known as the largest family of enzymes that contribute to both primary metabolism and the chemical diversity of plant secondary metabolites. It is important to elucidate the in vivo role of CYPs in secondary metabolism, in order to apply them in the production of valuable metabolites in medicinal plants via metabolic engineering. CYP76AH1 has been suggested to catalyze the conversion of the carbon skeleton miltiradiene into the intermediate ferruginol, which is involved in the biosynthesis of tanshinones, the chief bioactive ingredients of Salvia miltiorrhiza. However, its role in planta remains to be elucidated. In this work, we constructed a CYP76AH1 RNAi system for hairy roots. Metabolic analysis of RNAi-AH1 hairy root lines showed a significantly increased accumulation of miltiradiene compared to the control lines. At the same time, the concentration of ferruginol decreased revealing the in vivo catalytic activity of CYP76AH1. The content of tanshinones decreased significantly after silencing of CYP76AH1, which verified its key role in the biosynthesis of tanshinones, and indicated that it could be used as a target for metabolic engineering., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Secondary metabolites from plants play key roles in human medicine and chemical industries. Due to limited accumulation of secondary metabolites in plants and their important roles, characterization of key enzymes involved in biosynthetic pathway will enable metabolic engineering or synthetic biology to improve or produce the compounds in plants or microorganisms, which provides an alternative for production of these valuable compounds. Salvia miltiorrhiza, containing tanshinones and phenolic acids as its active compounds, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. The biosynthetic analysis of secondary metabolites in S. miltiorrhiza has made great progress due to the successful genetic transformation system, simplified hairy roots system, and high-throughput sequencing. The cloned genes in S. miltiorrhiza had provided references for functional characterization of the post-modification steps involved in biosynthesis of tanshinones and phenolic acids, and further utilization of these steps in metabolic engineering. The strategies used in these studies could provide solid foundation for elucidation of biosynthetic pathways of diterpenoids and phenolic acids in other species. The present review systematically summarizes recent advances in biosynthetic pathway analysis of tanshinones and phenolic acids as well as synthetic biology and metabolic engineering applications of the rate-limiting genes involved in the secondary metabolism in S. miltiorrhiza.

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, a full-length cDNA sequence of SQS2 from S. miltiorrhiza f. alba was cloned by the method of reverse transcription polymerase chain reaction (RT-PCR). The SmSQS2 cDNA sequence was obtained, this sequence is named SmSQS2 and its GenBank registration number is KM244731. The full length of SmSQS2 cDNA was 1245 bp, encoding 414 amino acids including 5'UTR 115 bp and 3'UTR 237 bp. Sequence alignment and phylogenetic analysis demonstrated that SmSQS2 had relative close relationship to the SQS2 of S. miltiorrhiza. The induction of E. coli [pET28-SQS2] in different temperature, induction time, IPTG concentrations and density of inducing host bacterium (A600) were performed, Shaking the culture at 30 degrees C until the A600 is approximately 0.6 and add IPTG to final concentration of 0.2 mmol x L(-1), and then the optimal expression of SmSQS2 recombinant protein were accumulated after the induction time of 20 h. The research provided important base for the study of sterol and terpene biosynthesis of SQS2 in S. miltiorrhiza f. alba.

Tanshinones, the main bioactive compounds of Salvia miltiorrhiza, are the diterpenoid pigments, multiple genes were proved to be involved in their biosynthesis in plants. CYP76AH1 is the initial P450 gene in the tanshinones biosynthetic pathway, its function has been validated by yeast expression and in vitroenzymatic reaction. In order to clarify the function of CYP76AH1 in vivo, in this study, we constructedthe RNA interference of CYP7AH1 in S. miltiorrhiza hairy root. The RNA interference vector with a hairpin structure was constructed using the Gateway technology, and then the interference fragment was integrated into the genome of S. miltiorrhiza mediated by Agrobacterium rhizogenes. Several highly CYP76AH1 interference S. miltiorrhiza hairy roots were obtained for further analysis.

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction (RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium (A600). The optimal expression conditions of SmHDS were 30 degrees C until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol x L(-1), and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.

NAC transcription factors involved in plant growth and development, as well as responses to biotic and abiotic stress. RNAi Vectors for SmNAC transcription factors of Salvia miltiorrhiza was constructed by using Gateway cloning technology, in order to further study the function of SmNAC1 transcription factor. According to Gateway cloning technology, the specific fragments of SmNAC1 containing attB adapter was amplified by PCR using ultra-fideling phusion polymerase of NEB. By the BP recombination reaction, the PCR product containing attB was transferred to an donor vector (pENTR/SD/D-TOPO). Finally, SmNACi specific gene was cloned into pK7GWIWG2D plant expression vectors by LR recombination reaction. Experimental results showed that Gateway cloning technology provide a rapid and highly efficient way to clone the interested gene.

Objective: To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system., Method: The 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI., Result: Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%., Conclusion: Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

Bioactive natural products are the material bases of Chinese materia medica resources. With successful applications of synthetic biology strategies to the researches and productions of taxol, artemisinin and tanshinone, etc, the potential ability of synthetic biology in the sustainable utilization of Chinese materia medica resources has been attracted by many researchers. This paper reviews the development of synthetic biology, the opportunities of sustainable utilization of Chinese materia medica resources, and the progress of synthetic biology applied to the researches of bioactive natural products. Furthermore, this paper also analyzes how to apply synthetic biology to sustainable utilization of Chinese materia medica resources and what the crucial factors are. Production of bioactive natural products with synthetic biology strategies will become a significant approach for the sustainable utilization of Chinese materia medica resources.

Salvia miltiorrhiza is a highly valued traditional chinese medicine for the treatment of atherosclerosis-related disorders in china, such as cardiovascular and cerebrovascular diseases in China. The wilt disease is serious in the culture of S. miltiorrhiza. Wilt disease cause biomass of plant shoots and roots is lessened, active components are decreased. To solve these problems, we research the pathogen causing wilt disease of S. miltiorrhiza. The suspected pathogen is identified by morphology and etiological test. The identification was further confirmed by alignment the sequences of internal transcribed spacer (ITS) amplified by PCR. Our result show the wilt disease of S. miltiorrhiza mostly occurred in July and August, which is hot and wetter. The wilt disease rate of S. miltiorrhiza continuous cropping for one year in S. miltiorrhiz stubble is 10%, but the wilt disease rate of S. miltiorrhiza continuous cropping for three years in S. miltiorrhiz stubble is 60%-70%. The root rot of S. miltiorrhiz caused by the wilt disease, so the wilt disease was mistaken for the rot root in production. Morphological characteristics show the pathogen is Fusarium oxysporum. The sequence of ITS wes determined and found by BLAST shared 99% identity to that of F. oxysporum f. sp. cucumerinum. So it comes to the conclusion that the causing agent of wilt disease on S. miltiorrhiza belongs to F. oxysporum.

Fosmidomycin (100 micromol x L(-1)) which is the effective inhibitor of DXR, key enzyme in terpenoid MEP pathway, was used to treat with hairy roots of Salvia miltiorrhiza. The treated roots were harvested at 2, 4, 6, 8, 10, 16 and 21 d, mRNA level of SmDXR and tanshinone content in treated and negative control groups were detected. Results found that, after treated with fosmidomycin, color of S. miltiorrhiza hairy roots grew pale gradually comparing with controls; mRNA level of SmDXR in hairy roots varied as a shape of parabolic and the highest value achieved at the sixth day after treatment, then it decreased gradually; Content of four kinds of tanshinones were detected. Among of the four kinds of tanshinones, Tanshinone I content changed relatively little, while content of dihydrotanshinone I, cryptotanshinone and tanshinone II (A) decreased gradually in 21 days. The content of total tanshinones in NC groups was 5, 63 times more than FOS-treated roots in the 21th day. The previous results showed that SmDXR played an important role in the accumulation of tanshinone content in MEP pathway. Once the mRNA level of SmDXR was suppressed, the accumulation of secondary metabolites will be significantly affected.

Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.

In order to study function of NAC transcription in development, hormone regulation and the stress response of Salvia miltiorrhiza, the NAC transcription was cloned and analyzed. By retrieving cDNA database of S. miltiorrhiza hairy root one NAC unigene was found, then a full length of cDNA was cloned by designing specific primers and PCR amplifying. Using ORF finder it was found that the cDNA containing a NAC-AB conserved domain in N-terminal, so the cDNA was a NAC transcription factor, named as SmNAC1 (kF006346). Bioinformatics analysis showed that SmNAC1 had an open reading frame (ORF) of 591 bp encoding 196 amino acids. The calculated protein had isoelectric point (pI) of 4.36 with molecular weight about 21.66 kDa. The transcription level of SmNAC1 after dealing with yeast extract (YE) and silver ion (Ag+) in S. miltiorrhiza hairy root was markedly stimulated up regulating. It was 1.4 fold compared with the control after induction 2 h, and maintained 2.0 fold on 4-12 h after induction. SmNAC1 may participate in regulation of stress response of YE + Ag+.

Chorismate mutase catalyzes the conversion of chorismate to prephenate that is the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine. A chorismate mutase gene, designated SmCM1, was isolated from Salvia miltiorrhiza by using RT-PCR. The full length of SmCM1 cDNA consists of 948 nucleotides and has an open reading frame of 765 bp. The deduced amino acid sequence of SmCM1 has 255 amino acid residues which forms a 36.0 kD polypeptide with calculated pI of 6.41 as expected. The putative polypeptide contains a CM_2 super family function domain. Blast W results showed that SmCM1 had 70% of the similarity with Petunia x hybrid CM, 72% of the similarity with Arabidopsis thaliana CM, and 64% of similarity with Populus trichocarpa CM. The transcription level of SmCM1 in root, stem and leaf was analysed by realtime quantitative PCR. The results showed the expression level of the SmCM1 in leaf was highest, and lowest in root. Yeast extract and silver ion joint induction could markedly stimulate the increase of mRNA expression of SmCM1 and its upstream 3-deoxy-7- phosphoheptulonate synthase (DAHPS) and chorismate synthase (CS). It was 7.9, 5.5 and 9.8 times of control on 8 h after induction, respectively.

Objective: A SmERF1 gene was isolated from Salvia miltiorrhiza, and expression patterns to different stress condition were analysed in the root tissues of S. miltiorrhiza., Method: The cDNA of SmERF1 gene from S. miltiorrhiza was isolated by RTPCR, and the phylogenetic tree using the neighbour-joining tree method in Mega 5 was obtained. To confirm the protein is likely to localize in the nucleus, the SmERF1 coding region was fused to the N-terminus of the GFP gene under the control of the CaMV 35S promoter and transferred into onion epidermal cells using the particle bombardment method. Semi-quantitative RT-PCR analysis revealed different expression pattern of SmERF1 gene in response to exogenous ABA, MeJA and SA., Result: The phylogenetic tree analysis revealed that SmERF1 is most similar to AP2/ERF VII subgroup members. The transient expression of the SmERF1::GFP fusion protein indicated that the SmERF1 was exclusively localized to the nucleus. The transcript of SmERF1 highly accumulated when the plants were treated with MeJA, while accumulated slightly in response to exogenous ABA, salicylic acid., Conclusion: These results suggest hormone such as ABA, MeJA and SA signaling pathways can be involved in the activation and inhibition of the SmERF1.

This study reported the obtainment of the full-length cDNA of Salvia miltiorrhiza hairy roots (Abbr: SmHDR, GenBank number: JX233817), via extracting Salvia miltiorrhiza hairy roots total RNA, designing specific primers according to the transcriptome data and using the RACE strategy, and then analyzed it with bioinformatics approaches. On this basis, using the real-time PCR to detect SmHDR gene expression after Ag+ induction, and testing tanshinones contents of corresponding samples by UPLC. SmHDR has 1 647 nucleotides, and an open reading frame (ORF) encoding a protein of 463 amino acid residues. The deduced protein has isoelectric point (pI) of 5.72 and a calculated molecular weight about 51.88 kD. In the secondary structure, the percentage of alpha helix, beta turn and random coil were 35.64%, 20.30% and 44.06%, respectively. Sequence alignment and phylogenetic analysis demonstrated that SmHDR had relative close relationship to the HDR of Picrorhiza kurrooa, similar to HDR from other species of plants. Real time PCR results indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmHDR. At the same time, results of ultra performance liquid chromatography (UPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy roots of Salvia miltiorrhiza were increased dramatically at 12 h after treated with Ag+, and then decreased significantly. This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by Ag+. The content of tanshinones was gradually raised, and it had an obvious increase at 120 h. The bioinformatics analysis and gene expression indicated that SmHDR might be involved in tanshinones biosynthesis, which laid the foundation for further study of secondary metabolic regulation mechanism of tanshinones.

Objective: Accumulation of tanshinton in Salvia miltiorrhiza are enhanced by exogenous application of jasmonates. The core JA signaling module COI1/JAZ/MYC2 play a central role on control of downstream gene expression in the JA pathway. To obtained the antibody of SmJAZ, SmJAZ recombinant protein was expressed in Escherichia coli and optimal expression was performed., Method: The full-length SmJAZ1 ORF was sub-cloned in a prokaryotic expression vector pET32a. The recombinant fusion protein had high expression level in BL21 (DE3) strain of E. coli, and SDS-PAGE analysis showed its molecular weight was about 24 kDa., Result: The induction of E. coli [pET32-JAZ1] in different temperature, induction time, IPTG concentrations and IPTG adding time of E. coli were performed. The induction time and the induction temperature are positively related trends with SmJAZ1 protein expression, and IPTG concentration had no significant impact in protein expression, whereas IPTG adding time had significant impact on protein expression., Conclusion: Shaking the culture at 30 degrees C until the A600 is approximately 0.9 (2 h in LB), and add IPTG to a final concentration of 0.1 mmol x L(-1), and then the optimal expression of SmJAZ1 recombinant protein were accumulated after the induction time of 20 h.

Objective: To clone and analysis a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (SmMCS) full-length eDNA from Salvia miltiorrhiza hairy roots., Method: A full-length eDNA of SmMCS has been cloned by designing specific primers according to the transcriptome database and using the RACE strategy. ORF Finder was used to find the open reading frame of SmMCS cDNA and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5. 1. Real-time quantitative PCR have been applied to detect the transcription level of SmMCS from hairy roots after elicitor Ag+ supplied., Result: The SmMCS cDNA sequence was obtained. The full length of SmMCS (DNA was 988 bp encoding 234 amino acids. The deduced protein had isoelectric point (pI) of 8.53 and a calculated molecular weight about 24. 6 kDa. Results of real time PCR indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmMCS in hairy roots, and were increased dramatically at 12 h., Conclusion: The full-length cDNA of SmMCS was cloned from S. miltiorrhiza hairy root,which can provide a gene target for further studies of tanshinones biosynthesis and terpenoid secondary metabolites.

The transcription factor of ethylene responsive factor binding protein (ERF) is belonged to AP2/ERF superfamily, which is known to be unique in plants. AP2/ERF proteins have important functions in the transcriptional regulation of a variety of biological processes related to growth and development, as well as various responses to environmental stimuli. An ERF gene from Salvia miltiorrhiza is cloned and divided into ERF gene family group VII of Arabidopsis and Rice. It contains a MCGGAI (I/L) motif referred to as CMVII-1 and a single intron in the 5'-flanking region of the AP2/ERF domain. Sequence analysis reveals that the region of second extron has abundant polymorphism sites. There are 21 single nucleotide polymorphism sites (SNPs) in the 264 bp region, among them, 14 SNPs are synonymous substitutions and 7 SNPs are non-synonymous substitutions. Though analysis of 181 samples from Shandong, Shaanxi and Sichuan Provinces, it reveals that each production area has its own special genotypes, 5 SNPs show significant difference. Cluster based on UPGMA method reveals that different populations from specific province have clustered together. It shows that SmERF gene will be a candidate molecular marker for the identification of Salvia miltiorrhiza from different areas.

Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpene synthesis pathway, catalyzed two units of acetyl-CoA to acetoacetyl-CoA. In order to study the tanshinone biosynthesis in Salvia miltiorrhiza, a novel AACT gene, SmAACT, was cloned using cDNA microarray and RACE strategy. The full length cDNA of SmAACT is 1 623 bp (accession No. EF635969), which contained a 1 200 bp open reading frame (ORF) encoding a 399 amino acid protein. Nine introns were found in the genomic sequence. SmAACT was upregulated by YE and Ag+ elicitors both with cDNA microarray and quantitative RT-PCR analyses along with the accumulation of tanshinones. Sequence homology comparison and phylogenetic analysis all suggested that SmAACT belonged to the class of acetyl-CoA C-acetyltransferase. The transcription level of SmAACT was relatively higher in root than that in stem and leaf tissues. SNP analysis revealed that SmAACT was highly variable in the region of 6 to 9 introns with 33 SNPs in the 600 bp region, there are 5 SNPs in the cDNA region while they are all synonymous cSNPs. Some special genotypes were found in Salvia miltiorrhiza from different areas. SmAACT will be an useful gene for further analyze the mechanism of gene regulation among the tanshinones biosynthesis.

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.

Objective: To study on the chemical stability of Salvia miltirrhiza hairy root., Method: The rolA gene was detected by PCR in DNA and the chemical contituent variances were detected by HPLC., Result: The rolA gene was found in all the 10 batches of the culfured hairy root. The similarities of the chromatographic fingerprints of the 10 batches are higher than 0. 95., Conclusion: There are no significant differences of the chemical constituents in 10 hairy root samples.

Objective: Studying the gene expression profiling of different stage hairy root of Salvia miltiorrhiza, in order to find functional genes., Method: The contents of second metabolites were determined by HPLC and gene expression profiling was detected by cDNA microarray. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine's linear RNA amplification method and subsequent enzymatic reaction. The microarrays were scanned with a ScanArray Express scanner using ScanArray 2.0 software and quantified by signal intensities of individual spots from the 16-bit TIFF images using GenePix Pro 4.0. The linear normalization method was used for data analyze. Northern blot was used to test the gene expression results obtained by microarray. Different expressed genes were sequenced and analyzed by gap4 software, and then they were analyzed with BLASTX, BLASTN, GO and KEGG., Result: Growth rate and second metabolites analysis indicated that the stage from 30 d to 45 d was the growth stage, while the stage from 45 d to 60 d was the second metabolites accumulation stage. Accordingly 30 d hairy root was chosen as a reference, which was hybridized with 45 d and 60 d hairy root separately. Total 203 different expressed genes were obtained. Northern blot showed that the result was identical with the microarray result. After sequenced, there were 172 genes clustered into 114 clusters (Unigenes). Among them, 62 unigenes had known functions, 34 unigenes were hypothetical protein, 9 unigenes were homologues with no similarity and 9 unigenes were unidentified protein with low similarity. Total 67 genes were classified into cellular component ontology, molecular function ontology and biological process ontology based on GO analysis. Total 26 genes, which represented 29 metabolic-related enzymes, were located in metabolic maps based on KEGG pathway classification., Conclusion: Several important functional genes related to second metabolite synthesis were cloned such as P450 and copalyl diphosphate synthase genes. cDNA microarray was a useful tool for functional genomics of traditional Chinese medicine.

Objective: To study the metallothionein genes of Salvia miltiorrhiza through bioinformatics and characterization of tissue expression in regenerated shoots., Method: Metallothionein genes were obtained by cDNA microarray analyze. BLAST was used for align, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT-PCR method was used to detect the gene expression level., Result: Two metallothionein genes were obtained which were contained a deduced amino acid sequence of 80 and 79 residues, named as SmMT-2a and SmMT-2b, they had a homology of 71.25%. Semiquantitative RT-PCR indicated that metallothionein genes were expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in leaf than in root and stem., Conclusion: It was the first time that metallothionein genes were obtained from S. miltiorrhiza. It provides a good basis for further functional study of S. miltiorrhiza.

Objective: Establishing cDNA microarray, in order to study functional genomics of Salvia miltiorrhiza., Method: Total RNA samples were prepared from S. miltiorrhiza roots using a modified CTAB method. mRNA was isolated by Quichprep Micro mRNA Purification Kit from Pharmacia. Then cDNA was synthesized and cloned into the EcoRI-XhoI sites of the ZAP Express vector using a cDNA synthesis kit, and the ligation mixture was packaged using a ZAP-cDNA Gigapack Gold III cloning kit (Stratagene). The single phage was isolated for PCR amplification, Aliquots of the PCR reactions were analyzed in a 1.5% agarose gel to verify the quality of PCR. The remaining cDNA was purified by Multiscreen filter plates (Millipore) and aliquots were analyzed by agarose gel again to verify the quality of purification. Clones passed verification was resuspended in 15 microL 50% DMSO for arraying. An actin gene from S. miltiorrhiza was used for positive control. PloyA and 50% DMSO was used for negative controls., Result: Bacterial colonies containing cNDAs of S. miltiorrhiza were inserted with average insert size of 0. 5 kb to 2. 5 kb. Total 4 354 genes were singled out from the first 8 736 PCR product and used for cDNA microarray manufacture. Single color fluorescence hybridization showed that all positive controls had signals while negative controls had no signals., Conclusion: It was the first cDNA microarray about traditional Chinese herbs especially for geoherbs. It could be a powerful tool for studying functional genomics of S. miltiorrhiza.

Objective: To study the effects of methyl jasmonate (MJ) on the accumulation and release of tanshinones in suspension cultures of Salvia miltiorrhiza hairy roots., Method: 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., Result: 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., Conclusion: MJ could stimulate the accumulation of tanshinones in S. miltiorrhiza and have released them into the culture medium.

Objective: To study the culture of adventitious root of Salvia miltiorrhiza in vitro systemically., Method: Effects of sucrose concentrations, medium pH, inoculum size and plant growth regulators on adventitious root growth and secondary metabolites production in S. miltiorrhiza were investigated., Result: With the increase of initial sucrose concentration, adventitious root growth rates increased and tanshinone II A content decreased, while content of protocatechuic aldehyde showed a broken line change and its highest production was obtained under 30 g x L(-1) sucrose in the medium. As for the effect of medium pH, medium pH of 6.5, 5.5 (or 6.0) and 5.8 was favorable for adventitious root growth, tanshinone II A and protocatechuic aldehyde synthesis respectively. Furthermore, adventitious root growth, rate was greatly increased when inoculum size was 2.5%. MS medium added with 0.5 mg x L(-1) KT was much favorable for tanshinone II A and protocatechuic aldehyde accumulation., Conclusion: Parameters including sucrose concentrations, medium pH, inoculum size and plant growth regulators have distinct effects on the in vitro culture of adventitious root growth and secondary metabolites synthesis of S. miltiorrhiza.

Objective: To study the effects of mineral cations on the growth of Salvia niltiorrhiza adventitious roots and the accumulation of tanshinone II A and protocatechuic aldehyde., Method: The adventitious roots were cultured under different concentrations of metal cations and the contents of tanshinone II A and protocatechuic aldehyde were determined by HPLC., Result: Fe2 + and Mn2+ in MS medium are suitable for adventitious root growth, but Cu2+ and Mg2+ in high concentration could improve root proliferation, and Zn2+ has no obvious effect on root growth. As for tanshinone II A and protocatechuic aldehyde biosynthesis, Cu2+ and Zn2+ would inhibit the biosynthesis of protocatechuic aldehyde, furthermore, proper Fe2+ and Mg2+ concentration could advance the biosynthesis of protocatechuic aldehyde, whereas, Mn2+ in high concentration could accelerate the biosynthesis of protocatechuic aldehyde. Cu2+, Zn2+, Mg2+ in low concentration and Fe2+ and Mn2+ in high concentration would all advance the biosynthesis of tanshinone II A., Conclusion: The mineral cations have obvious effects on the secondary metabolites biosynthesis in adventitious root culture of S. niltiorrhiza.