Cloning Of Genes Involved In Tanshinones Biosynthesis And Its Metabolic Regulation

Cardiovascular disease and cerebral vascular disease are the top killers that threat human heath and life. Salvia miltiorrhiza is a well-known and very important traditional Chinese medicinal herb. As the major active constituents of S.miltiorrhiza, tanshinones are the important medical resources for the treatment of Cardiovascular disease and Cerebral vascular disease in clinical, recently studies also indicated that tanshinones had anti-tumor potential. Therefore, S.miltiorrhiza has been very widespread used in clinical. However, due to the limited available resources of S.miltiorrhiza and low content of bioactive components in S.miltiorrhiza, the application of tanshinone was restricted.Herein, the cloning and characterization of full-length cDNAs encoding geranylgeranyl diphosphate synthase (SmGGPPS, GenBank Accession No.FJ643617) and 3-hydroxy-3-methylglutaryl-CoA reductase (SmHMGR, GenBank Accession No.EU680958) from S.miltiorrhiza by rapid amplification of cDNA ends (RACE) was investigated. The function of SmGGPPS was also confirmed by color complementation assay in E. coli DH5α.The full-length cDNA of SmHMGR was 2115 bp containing a 1695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyses revealed that the deduced SmHMGR had extensive homology with other plant HMGRs containing two transmembrane domains and a catalytic domain. 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.The full-length cDNA of SmGGPPS was 1234 bp containing a 1092 bp open reading frame (ORF) encoding a polypeptide of 364 amino acids. Analysis of SmGGPPS genomic DNA revealed that it contained two exons and one intron. Bioinformatic analyses revealed that the deduced SmGGPPS had extensive homology with other plant GGPPSs contained all five conserved domains and functional aspartate-rich motifs of the prenyltransferases. 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 three tested tissues, suggesting that SmGGPPS was elicitor-responsive.The work further analyzed the relationship between the expressions of genes involving in tanshinones biosynthesis pathway (SmHMGR, SmGGPPS, SmDXR, SmAACT, SmCMK, SmIPPI, SmFPPS, SmCPS, SmKSL) and tanshinones accumulation in S.miltiorrhiza hairy roots under the treatment of MJ,Ag+,YE and YE+Ag+ respectively by RT-PCR and HPLC. Our results showed a tight correlation between gene expression and tanshinone accumulation, suggesting that tanshinone accumulation may be the result of the co-expression up-regulation of several genes involved in tanshinone biosynthesis under treatment of various elicitors. Meantime, SmHMGR, SmDXS2, SmFPPS, SmGGPPS and SmCPS were identified as the potential key enzymes in the pathway for targeted metabolic engineering to increase accumulation of tanshinone in S. miltiorrhiza hairy roots. This work provides useful information to further understand molecular regulation mechanism of genes encoding related enzymes involved in tanshinones biosynthesis.