Improving Tanshinone Accumulation In Salvia Miltiorrhiza Hairy Roots By The Strategy Of Co-expressing Double Genes

Dan shen (Salvia miltiorrhiza Bunge) is a famous traditional Chinese herb and has been extensively used to treat many kinds of cardiovascular diseases in clinical. Tanshinone (including cryptotanshinone, tanshinone I, tanshinone IIA, etc), which is one kind of major lipid-soluble active constituent of Salvia miltiorrhiza, has been shared various pharmacological activities.3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and geranylgeranyl diphosphate synthase (GGPPS) were two key catalyzing enzymes in the tanshinone biosynthesis pathways. Here, the cDNAs of HMGR and GGPPS from S. miltiorrhiza, driven by a CaMV 35S promoter in pCAMBIA1304+, were separately or simultaneously introduced into S. miltiorrhiza firstly and hairy roots were obtained by Agrobacterium rhizogenes infection. PCR and RT-PCR were used to detect the integration and expression of SmHMGR and SmGGPPS, and the tanshinone production of transformed hairy roots was determined by HPLC. The results showed that overexpression of SmHMGR can lead to enhanced tanshinone production and the highest content of tanshinone was detected in H12 (1.530 mg/g DW, 2.2 fold higher than the control), which suggested that MVA pathway also play an important role in diterpene biosynthesis at least in S. miltiorrhiza. G50 (2.477 mg/g DW, 4.2 fold higher than the control) was detected as the highest tanshinone production in hairy roots overexpression of SmGGPPS. It showed that SmGGPPS exhibited a more powerful pulling effect than did SmHMGR, which may be due to SmGGPPS is closer in the reaction pathway to tanshinone. Combination use of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g DW, 4.7 fold higher than the control) in line HG9, which showed that co-expression of SmHMGR and SmGGPPS can produce a positive cooperative effect on stimulating tanshinone accumulation. This is the first report on enhancement of tanshinone production in S. miltiorrhiza by means of co-expression of SmHMGR and SmGGPPS, which is a major breakthrough for studying metabolic engineering of tanshinone.