| The root of Salvia miltiorrhiza Bunge (Danshen) is the traditional medicine with the function of invigorating the circulation of blood and removing blood stasis, and recently was found to remarkably affect in the treatment of cardiovascular and cerebrovascular diseases. It contains two kinds of biologically active compounds, water-soluble poly-phenolic compounds and fat-soluble compounds , including tanshinones. Because of having the functions of natural anti-oxidation, anti-atherosis, antibacterial and anti-tumor, the Chinese herb is more popular all of the world. However, the wild resources of Danshen have been disrupted by excess picking, and the quality of cultivars degenerates and intermingles, so in order to radically solve these problems, to industrializedly product the active compenents of Danshen, and to well exploit the herb, the biosynthesis pathway must be elucidated. It has been known that tanshinones are the diterpenoid derivatives, like other diterpenoids, they come from the isoprenoid metabolic pathway, and the precursor is geranylgeranyl pyrophosphate (GGPP), but the detail process since GGPP is still unclear. GGPP is the product of the condensation of isoprenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP, C15), catalyzed by GGPP synthase. It is the universal precursor of diterpenoids, providing the framework of other diterpenoids, carotenoid, chlorophyll, quinine and the side of vitamin E. Because GGPP is the common precursor of primary and secondary metabolites, GGPP synthase can regulate the flux of carbon, and then become one of the key enzymes among the primary and secondary metabolism pathways.GGPS is ubiquitous in plants, animals and bacteria. It has the well conservative sequences, and the phylogenetic analysis shows the proteins have the evident phyletic specific, and the GGPS of animals, plants, bacteria, fungi and archaea come from the different branchs. The studies of GGPS in the model plant, Arabidopsis thaliana, show that there are several GGPS genes in plants, transporting to the different organelles, expressing temporally and spacially, so the functions in metabolic pathways are distinct. It has been known GGPP is also the precursor of the taxol and ginkgolide which are officinal chemicals, and it has been proved that GGPS is one of the key enzymes in the synthesis pathway of taxols in yew.The degenerate primers were designed based on the conservative regions, the first aspartae-rich motif and the second aspartate-rich motif, of GGPS protein sequences from public databases. The core target fragment of about 400bp was obtained from Danshen root by use of homologous cDNA amplification, and then the sides of the core fragment were complemented with the RACE technology, finally the 1 298bp and 1 177bp cDNA were obtained, respectively named SmGGPS1 and SmGGPS2. Bioinformatics analysis showed that the two sequences have respectively 1 095bp and 1035bp ORF, more than 60% identity to the reported plant GGPS genes; they were deduced to encode 364 and 344 amino acids, the molecular weight of the two proteins are respectively 38 986.6 and 37 373.4, pI are 6.29 and 6.78, and the polyprenyl synthetase signature is both found. By TargetP 1.1, it is predicted that SmGGPS1 has a plastid targeting signal peptide of about 52 amino acid at the N-terminal end, and SmGGPS2 has a mitochondrion targeting signal peptide of about 19 amino acid at the N-terminal end. RT-PCR semi-quantitative analysis showed that the two genes expresse in the all tested tissues, and SmGGPS1 with much higher level of expression in the leaves in the flowering stage, SmGGPS2 with much higher level of expression in the roots and flowers in the flowering stage. SmGGPS1 has a 397 bp intron, and SmGGPS2 has no intron.By the Gateway technology, the vectors expressed in yeast was constructed, and transformed to SFNY368 that is sensitive to lower temperature because of the absent GGPS. According to the functional complement, it was validated that SmGGPS1 encodes the functional GGPP synthase, while SmGGPS2 cannot well complement to the function of SFNY368.It was explained that the signal peptide of SmGGPS2 influenced its expression in yeast, and it must be validated further.For SmGGPS1, RNAi vectors, overexpression vector and the two control vectors were constructed. With RNAi vectors, for the first time, Agrobacterium rhizogenes-mediated transformation of Salvia miltiorrhiza Bunge was performed, brought the transgenetic hairy root with the two screening markers of antibiotic and GFP, and the induction rate and the average transformation rate were both more than 50%. Taking use of the transforming method, the SmGGPS1 overexpression and the control transgenetic hairy root were obtained, then 20 lines overexpression transgenetic hairy root and 17 lines control transgenetic hairy root were cultured in liquid culture medium. After 45 days, the compounds were extracted by the metabolism methods. The results of analysis by HPLC showed the ratios of the lines of overexpression transgenetic hairy root and control transgenetic hairy root containing tanshanone IIA are respectively 90% and 100%, containing tanshanone I all 35%, containing cryptotanshinone respectively 20% and 71%. On the distribution of all tanshanones, in the control transgenetic hairy roots, it showed basically normal distribution, while in overexpression transgenetic hairy roots, it didn't show distinct, except NO.15. The result showed that the over expression of SmGGPS1 induced the sharp decrease of cryptotanshinone content, and then the lower of all tanshanones content. 19 compounds detected by HPLC were selected and analysized by PCA, the result showed the components of overexpression lines and control lines can be apart, proving that over-expression group of compounds tested is not a random event, but the overall characteristics by cause of the expression level of SmGGPS1 gene change; and at the same time, five metabolic markers were found. The cluster result of chemicals showed the nineteen components of chosen can be classed to three sort, and two of them might be correlative with the synthesis of tanshinones. By Agrobacterium tumefaciens-mediated method, 32 lines of RNAi2, 10 lines of RNAi3, and 41 lines of pDONR(-) transgenetic plants were obtained, and 8 lines of RNAi2 and 1 lines of RNAi3 ones exhibited macroscopicly the reduced roots and yellow leafs, which hinted the increased content of gibberellin and the decreased content of chlorophyll and (or) carotenoid.Conclusion: For the first time, the two GGPS genes of Salvia miltiorrhiza Bunge were obtained, and then the function of SmGGPS1 was confirmed with yeast complement test. And firstly, the Agrobacterium rhizogene-transformed transgenetic method to Danshen was established. From the type relationship of overexpreesion transgenetic hairy roots and RNAi transgenetic plants, it can be concluded that one of the function of SmGGPS1 is to synthesize chlorophyll and (or) carotenoid, but not gibberellin and tanshinones. At the same time, it can be presumed that the biosynthesis pathway of gibberellin and tanshinones shares several steps. |
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