Molecular Cloning And Expression Analyses Of Genes Involved In Tanshinones Biosynthesis In Salvia Miltiorrhiza

Danshen, first recorded in the Shen long's herbal classic, was a famous traditional Chinese medicinal herb, which has effects such as dissolving stasis and odynolysis, blood-activating and regulating of the menstrual function, nourishing the heart and relieving restlessness. The active ingredients of S.miltiorrhiza were fat-soluble diterpene quinine compounds and water-soluble phenolic acid ones, both of which had strong pharmacological activity. The diterpene quinine compounds, such as Tanshinone IIA, were widely applied in bacteriostasis, diminishing inflammation, oxidation resistance and anticancer et al, so the clinical requirement is tremendous.However, because of the long growth cycle(more than two years), there were many defects in the S.miltiorrhiza's traditional culture, such as the degeneration of quality, and the relative large cost of production;there were also many defects in the chemosynthesis, such as the complicated course, the huge cost, the environment pollution; the active ingredients production were very low and unstable in cell culture. The modern genetic engineering technologies were used to put the key-enzyme of the metabolic pathway into the S.miltiorrhiza's genome and then the transgenetic hairy root, transgenetic cell line and the regeneration plant were gained and to be cultivated on a large scale. These methods can provide us a new way to produce tanshinones and this way can be considered as an optimal path to resolve the problem such as the low production of tanshinones and the the shortage of the S.miltiorrhiza's resources.Until now, there are few researches and reports of the molecular biology about the biosynthesis of S.miltiorrhiza's secondary metabolites. So the S.miltiorrhiza was used as experimental material, RACE was used as technology platform and the homologous clone used as experimental considerations in this experiment. Three genes, involved the tanshinones'biosynthesis, were first cloned and characterized, including: 1-deoxy-d-xylulose 5-phosphate synthaseI, 1-deoxy-d-xylulose 5-phosphate synthaseII, 3-hydroxy-3-methylglutaryl-coenzyme A. The Tissue expression profile analysis and the expression profile analysis treated with the elicitors were also tested in this experiment. The results obtained were showed as below.1. A new full-length cDNA (2519bp) encoding 1-deoxy-d-xylulose 5-phosphate synthase I (designated as SmDXS I) was successfully isolated from S.miltiorrhiza by rapid amplification of cDNA ends (RACE). The full-length cDNA containd a 2145bp ORF (Open Reading Frame), encoding a deduced protein of 714 amino acid residues. Amino acid sequence comparison analysis showed that SmDXS I had high similarity with DXSI from other reported plants. Squence analysis showed that this protein contained a TPP banding domain(thiamin pyrophosphate binding domain), a conserved (DRAG domain)and a conserved His. Tissue expression profile analysis showed that SmDXS1 was detected in all tested tissues but at different levels with the highest expression in leaves, followed by in stems and roots, so it was considered to be a constitutively expressing gene. The Profile of SmDXSI under induction of elicitors showed that the elicitors treatments had few effect on the expression of SmDXSI which was not an elicitors-responsive gene.2. A new full-length cDNA (2522bp) encoding 1-deoxy-d-xylulose 5-phosphate synthase I (designated as SmDXSII) was successfully isolated from S.miltiorrhiza by rapid amplification of cDNA ends (RACE). The full-length cDNA containd a 2175bp ORF (Open Reading Frame), encoding a deduced protein of 724 amino acid residues. Amino acid sequence comparison analysis showed that SmDXSII had high similarity with DXSII from other reported plants. Squence analysis showed that this protein contained a TPP banding domain(thiamin pyrophosphate binding domain), a conserved (DRAG domain)and a conserved His. Tissue expression profile analysis showed that SmDXSII expression was weakly detected in roots and barely in stems and leaves, so it was considered to a non-constitutively expressing gene. The Profile of SmDXSII under induction of elicitors showed that SmDXSII was regulated in the transcription level at least; it was an elicitors-responsive gene, methyl jasmonate (MJ) and silver ion (Ag+) both can effectively up-regulated the expression of SmDXSII, but MJ's effects was better than Ag+.3. A new full-length cDNA (1655bp) encoding 3-hydroxy-3-methylglutaryl-CoA synthase (designated as SmHMGS) was successfully isolated from S.miltiorrhiza by rapid amplification of cDNA ends (RACE). The full-length cDNA containd a 1383bp ORF (Open Reading Frame), encoding a deduced protein of 460 amino acid residues. Amino acid sequence comparison analysis showed that SmHMGS had high similarity with HMGS from other reported plants. Tissue expression profile analysis showed that SmHMGS expression was detected in all tested tissues but at different levels with the highest expression in leaves, followed by in stems and roots, so it was considered to be a constitutively expressing gene. The profile of SmHMGS under induction of elicitors showed that SmHMGS was regulated in the transcription level at least; it was an elicitors-responsive gene, methyl jasmonate (MJ) and Yeast Extract (YE) both can effectively regulated the expression of SmHMGS.In this experiment, three genes involved in the tanshinones biosynthesis were isolated from S.miltiorrhiza, which can be very helpful for inhancing the tanshinones'production and S.miltiorrhiza's genetic improvement; Tissue expression pattern analyses of these cloned genes provides primary molecular evidence for further understanding of the genetic basis of tanshinones content diversity in different parts; expression profile analyses treated with the elicitors were carried out to provide useful information to further understand induction expression and molecular regulation mechanism of genes encoding related enzymes involved in tanshinones'biosynthesis.