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ABSTRACT: A new putative gene encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (designated as SmHMGS, GenBank Accession No. FJ785326), which catalyses the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA
as an early step in the mevalonic acid pathway, was isolated from young leaves of Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of the putative SmHMGS was 1,655bp containing a 1,381bp open reading frame (ORF) encoding a polypeptide of 460 amino acids. Comparative and bioinformatic
analyses revealed that SmHMGS showed extensive homology with HMGSs from other plant species. Phylogenetic tree analysis indicated
that SmHMGS belonged to the plant HMGS super family and had the closest relationship with HMGS from Hevea brasiliensis. Tissue expression pattern analysis revealed that the putative SmHMGS was constitutively expressed in all the tested tissues and strong in leaf, moderate in stem, weak in root, which was in contrast
to SmHMGR reported before. The putative SmHMGS was found to be an elicitor-responsive gene, which could be induced by exogenous elicitors, including salicylic acid (SA)
and methyl jasmonate (MJ). These results will help in understanding the role of HMGS in tanshinones biosynthesis in S. miltiorrhiza.
Keywords
Salvia miltiorrhiza
–Putative SmHMGS
–Expression profile–Salicylic acid (SA)–Methyl jasmonate (MJ)
Acta Physiologiae Plantarum 04/2012; 33(3):953-961. · 1.64 Impact Factor
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ABSTRACT: The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the
first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR
was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115bp containing a 1,695bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes
revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling
showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated
that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones
biosynthesis at the molecular level.
Acta Physiologiae Plantarum 04/2012; 31(3):565-572. · 1.64 Impact Factor
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ABSTRACT: 1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plants. The present study describes the cloning and characterization
of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255). Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with
DXRs from other plant species. Phylogenetic tree analysis indicated that SmDXR belongs to the plant DXR superfamily and has
the closest relationship with DXR from Lycopersicon esculentum. Tissue expression pattern analysis revealed that SmDXR expressed strongly in leaves, followed by roots and stems, implying that SmDXR was a constitutively expressed gene. This is the first report on the mRNA expression profile of genes encoding key enzymes
involved in tanshinone biosynthetic pathway in Salvia plants. The expression profiles revealed by RT-PCR under different elicitor treatments such as methyl jasmonate (MJ) and
salicylic acid (SA) were compared for the first time, and the results revealed that SmDXR was an elicitor-responsive gene, which could be induced by SA in leaves and inhibited by exogenous MJ in three tested tissues.
The functional color assay in Escherichia coli showed that SmDXR could accelerate the biosynthesis of lycopene, indicating that SmDXR encoded a functional protein. The
characterization, expression profile and functional analysis of SmDXR gene will be helpful for further study in the role of SmDXR in tanshinones biosynthetic pathway and metabolic engineering to increase tanshinones production in S. miltiorrhiza.
Acta Physiologiae Plantarum 04/2012; 31(5):1015-1022. · 1.64 Impact Factor
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ABSTRACT: Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP),
which is a key precursor for diterpenes including tanshinone. In this study, a full-length cDNA encoding GGPPS was isolated
from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmGGPPS (GenBank Accession No. FJ643617). The full-length cDNA of SmGGPPS was 1,234 bp containing a 1,092 bp open reading frame (ORF) encoding a polypeptide of 364 amino acids. Analysis of SmGGPPS genomic DNA revealed that it contained 2 exons and 1 intron. Bioinformatics analyses revealed that the deduced SmGGPPS had extensive homology with other plant GGPPSs contained all 5 conserved domains and functional aspartate-rich motifs of
the prenyltransferases. Molecular modeling showed that SmGGPPS is a new GGPPS with a spatial structure similar to other plant GGPPSs. Phylogenetic tree analysis indicated that SmGGPPS belongs to the plant GGPPS super-family and has the closest relationship with GGPPS from Nicotiana attenuate. The functional identification in Escherichia coli showed that SmGGPPS could accelerate the biosynthesis of carotenoid, demonstrating that SmGGPPS encoded a functional protein. Expression pattern analysis implied that SmGGPPS expressed higher in leaves and roots, weaker in stems. The expression of SmGGPPS could be up-regulated by Salicylic acid (SA) in leaves and inhibited by methyl jasmonate (MeJA) in 3 tested tissues, suggesting
that SmGGPPS was elicitor-responsive. This work will be helpful to understand more about the role of SmGGPPS involved in the tanshinones biosynthesis pathway and metabolic engineering to improve tanshiones production in S. miltiorrhiza.
KeywordsExpression analysis-functional identification-
Salvia miltiorrhiza
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SmGGPPS
-tanshinones
Biotechnology and Bioprocess Engineering 04/2012; 15(2):236-245. · 1.28 Impact Factor
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ABSTRACT: Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.
Metabolic Engineering 02/2011; 13(3):319-27. · 5.61 Impact Factor
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ABSTRACT: Salvia miltiorrhiza Bunge, called Dan shen in China, is a well-known traditional Chinese herb and has been used widely for the treatment of cardiovascular diseases due to its better performance and fewer side effects as confirmed in the long-time clinical use. Tanshinones, one class of lipid-soluble diterpene quinones from S. miltiorrhiza, were found to exhibit various pharmacological activities such as anti-oxidative, anti-inflammatory and anti-tumor properties. Due to the great importance of tanshinone and to its complex chemical nature as well as low content in plants, many efforts have been made to improve tanshinone production including hairy root culture, elicitor treatment and genetic engineering with new advance in recent years. Pharmacological activities of tanshinones and metabolic regulation of tanshinones biosynthesis in S. miltiorrhiza were reviewed in this paper.
Journal of Medicinal Plants Research. 01/2011; 4:2591-2597.
