1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.KM879187), 2148 bp (TwDXS2, GenBank accession no.KM879186), 1410 bp (TwDXR, GenBank accession no.KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g⁻¹, a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f.

Tanshinones are abietane-type norditerpenoid quinone natural products found in a well-known traditional Chinese medicinal herb, Salvia miltiorrhiza Bunge. The copalyl diphosphate synthase of S. miltiorrhiza (SmCPS) is the key enzyme in the first step for transformation of geranylgeranyl diphosphate (GGPP) into miltiradiene, which has recently been identified as the precursor of tanshinones. Based on previous gene-to-metabolite network, this study examined the influences of various combined elicitors on the expression of SmCPS and production of tanshinones in S. miltiorrhiza hairy root cultures. Combined elicitors were composed of three classes of elicitors, a heavy metal ion (Ag⁺), a polysaccharide (yeast extract, YE), and a plant response-signalling compound (methyl jasmonate, MJ). YE + Ag⁺, Ag⁺ + MJ, YE + MJ, and YE + Ag⁺ + MJ were the combinations we tested. The effect of elicitors on the SmCPS expression level was detected by quantitative real-time PCR (qRT-PCR), and the tanshinones accumulation responses to elicitation were analysed by Ultra Performance Liquid Chromatography (UPLC) metabolite profiling. Of these combined elicitors, the expression of SmCPS was significantly enhanced by elicitation, especially at 24 h and 36 h. Of four tanshinones detected, the contents of cryptotanshinone and dihydrotanshinone I were enhanced by treatment with YE + Ag⁺, Ag⁺ + MJ, and YE + Ag⁺ + MJ. Our results indicate that appropriate combined elicitors can enhance tanshinones production in hairy root cultures.

Tripterygium wilfordii is a traditional Chinese medical plant used to treat rheumatoid arthritis and cancer. The main bioactive compounds of the plant are diterpenoids and triterpenoids. 3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the reaction of acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, which is the first committed enzyme in the mevalonate (MVA) pathway. The sequence information of HMGS in Tripterygium wilfordii is a basic resource necessary for studying the terpenoids in the plant. In this paper, full-length cDNA encoding HMGS was isolated from Tripterygium wilfordii (abbreviated TwHMGS, GenBank accession number: KM978213). The full length of TwHMGS is 1814 bp, and the gene encodes a protein with 465 amino acids. Sequence comparison revealed that TwHMGS exhibits high similarity to HMGSs of other plants. The tissue expression patterns revealed that the expression level of TwHMGS is highest in the stems and lowest in the roots. Induced expression of TwHMGS can be induced by MeJA, and the expression level is highest 4 h after induction. The functional complement assays in the YML126C knockout yeast demonstrated that TwHMGS participates in yeast terpenoid biosynthesis.