Identification and functional characterization of squalene epoxidases and oxidosqualene cyclases from Tripterygium wilfordii.

KEY MESSAGE: We cloned two squalene epoxidases and five oxidosqualene cyclases, and identified their function using CRISPR/Cas9 tool and yeast heterologous expression. Triterpenes are the main active ingredients of Tripterygium wilfordii Hook.f., a traditional Chinese medicinal plant with many encouraging preclinical applications. However, the biosynthetic pathways of triterpenes in this plant are poorly understood. Here, we report on the isolation and identification of two squalene epoxidases (SQE6 and SQE7) and five oxidosqualene cyclases (OSC4-8) from T. wilfordii. Yeast complementation assays showed that TwSQE6 and TwSQE7 can functionally complement an erg1 yeast mutant that was constructed using the CRISPR/Cas9 system. The putative OSC genes were functionally characterized by heterologous expression in yeast. GC/MS analysis of the fermentation products of the transgenic yeast showed that both TwOSC4 and TwOSC6 are cycloartenol synthases, while TwOSC8 is a ß-amyrin synthase. The discovery of these genes expands our knowledge of key enzymes in triterpenoid biosynthesis, and provides additional target genes for increasing the production of triterpenes in T. wilfordii tissue cultures by disrupting competing pathways, or in chassis cells by reconstituting the triterpenoid biosynthetic pathway.

[Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii].

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 µmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of ß-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.

[Bioinformatics and tissue distribution analysis of Tripterygium wilfordii CYP450].

Cytochrome P450 family is a kind of biocatalyst widely existing in nature. It has many functions such as catalyzing the biosynthesis of plant secondary metabolites and regulating phytoremediation. Based on the analysis of proteome data of Tripterygium wilfordii,the CYP450 gene of T. wilfordii was preliminarily analyzed and predicted by various bioinformatics methods. The results showed that after the expression of T. wilfordii suspension cells was induced by methyl jasmonate,the proteomic data of T. wilfordii were obtained and analyzed,and 10 CYP450 proteins of T. wilfordii were finally screened out. By analyzing the phylogenetic tree constructed with CYP450 gene of Arabidopsis family,the 10 CYP450 proteins were clustered into 6 different CYP450 families. The physical and chemical properties of CYP450 proteins in different families were different. The secondary structure of CYP450 proteins was mainly composed of irregular curls. Eight subcellular localization results of CYP450 proteins were chloroplasts and the rest were plastids. Subsequently,the conserved domains( heme active sites) shared by CYP450 genes were found by analyzing the results of multiple sequence alignment. Finally,by analyzing the transcriptome data of T. wilfordii,the expression distribution of T. wilfordii in different tissues was preliminarily confirmed,which verified its correlation with the biosynthesis of active components of T. wilfordii,and provided important genetic resources for the analysis of biosynthesis pathway of active components of T. wilfordii.

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.

The gibberellin 13-oxidase that specifically converts gibberellin A9 to A20 in Tripterygium wilfordii is a 2-oxoglutarate-dependent dioxygenase.

MAIN CONCLUSION: A novel GA13-oxidase ofTripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase. It specifically catalyzes the conversion of GA9to GA20, but not GA4to GA1. Gibberellins (GAs) play essential roles in plant growth and development. Previous characterization of GA20- and GA3-oxidases yielded a large number of genetic elements that can interconvert different GAs. However, enzymes that catalyze the 13-hydroxylation step are rarely identified. Here, we report that the GA13-oxidase of Tripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase instead of reported cytochrome P450 oxygenases, among 376 differential proteins in comparative proteomics. Phylogenetic analysis showed that the enzyme resides in its own independent branch in the DOXC class. Unexpectedly, it specifically catalyzes the conversion of GA9 to GA20, but not GA4 to GA1. Contrary to the previous research, TwGA13ox transcriptional expression was upregulated ~ 146 times by exogenous application of methyl jasmonate (MeJA). RNAi targeting of TwGA13ox in T. wilfordii led to an 89.9% decrease of triptolide, a diterpenoid epoxide with extensive anti-inflammatory and anti-tumor properties. In subsequent MeJA supplementation experiments, triptolide production increased 13.4-times. TwGA13ox displayed root-specific expression. Our results provide a new GA13-oxidase from plants and elucidate the metabolic associations within the diterpenoid biosynthetic pathway (GAs, triptolide) at the genetic level.

Probing the function of protein farnesyltransferase in Tripterygium wilfordii.

KEY MESSAGE: We found two subunits FTase/GGTaseI-α and FTase-ß formed a heterodimer to transfer a farnesyl group from FPP to protein N-dansyl-GCVLS, confirming they are responsible for protein farnesylation in planta. Tripterygium wilfordii is a medicinal plant with a broad spectrum of anti-inflammatory, immunosuppressive and anti-cancer activities. Recently, a number of studies have focused on investigating the biosynthetic pathways of its bioactive compounds, whereas little attention has been paid to the enzymes which play important roles in regulating diverse developmental processes of T. wilfordii. In this study, we report for the first time the identification and characterization of two subunits of farnesyltransferase (FTase), farnesyltransferase/geranylgeranyltransferase I-α (TwFTase/GGTase I-α) and farnesyltransferase-ß (TwFTase-ß), in this important medicinal plant. Cell-free in vivo assays, yeast two-hybrid (Y2H) and pull-down assays showed that the two subunits interact with each other to form a heterodimer to perform the role of specifically transferring a farnesyl group from FPP to the CAAX-box protein N-dansyl-GCVLS. Furthermore, we discovered that the two subunits had the same cytoplasmic localization pattern and displayed the same tissue expression pattern. These results indicated that we identified a functional TwFTase enzyme which contains two functionally complementary subunits TwFTase/GGTase I-α and TwFTase-ß, which provides us promising genetic targets to construct transgenic plants or screen for more adaptable T. wilfordii mutants, which are able to survive in changing environments.

Functional characterization of squalene epoxidase genes in the medicinal plant Tripterygium wilfordii.

Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1-4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1-4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii.

Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii.

Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coliTransetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii.

Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii.

Tripterygium wilfordii, which has long been used as a medicinal plant, exhibits impressive and effective anti-inflammatory, immunosuppressive and anti-tumor activities. The main active ingredients are diterpenoids and triterpenoids, such as triptolide and celastrol, respectively. A major challenge to harnessing these natural products is that they are found in very low amounts in planta. Access has been further limited by the lack of knowledge regarding their underlying biosynthetic pathways, particularly for the abeo-abietane tri-epoxide lactone triptolide. Here suspension cell cultures of T. wilfordii were found to produce triptolide in an inducible fashion, with feeding studies indicating that miltiradiene is the relevant abietane olefin precursor. Subsequently, transcriptome data were used to identify eight putative (di)terpene synthases that were then characterized for their potential involvement in triptolide biosynthesis. This included not only biochemical studies which revealed the expected presence of class II diterpene cyclases that produce the intermediate copalyl diphosphate (CPP), along with the more surprising finding of an atypical class I (di)terpene synthase that acts on CPP to produce the abietane olefin miltiradiene, but also their subcellular localization and, critically, genetic analysis. In particular, RNA interference targeting either both of the CPP synthases, TwTPS7v2 and TwTPS9v2, or the subsequently acting miltiradiene synthase, TwTPS27v2, led to decreased production of triptolide. Importantly, these results then both confirm that miltiradiene is the relevant precursor and the relevance of the identified diterpene synthases, enabling future studies of the biosynthesis of this important bioactive natural product.

[Cloning and protein expression analysis of monoterpene synthase gene TwMS in Tripterygium wilfordii].

In this study, we cloned a monoterpene synthases, TwMS from Tripterygium wilfordii suspension cells. TwMS gene contained a 1 797 bp open reading frame (ORF), encoding a polypeptide of 579 amino acids, which deduced isoelectric point (pI) was 6.10 and the calculated molecular weight was 69.75 kDa. Bioinformation analysis showed that the sequence of TwMS was consistent with the feature of monoterpene synthases. Differential expression analysis revealed that the relative expression level of TwMS increased significantly after being induced by methyl jasmonate (MeJA). The highest expression level occurred at 24 h. TwMS protein was successfully expressed in Escherichia coli BL21 (DE3), which laid the foundation for identifying the function of T. wilfordii monoterpene synthases.

[Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii].

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

[Cloning and expression analysis of kaurenoic acid oxidase gene in Tripterygium wilfordii].

Kaurenoic acid oxidase involved in biosynthesis pathway of gibberellin. According to the transcriptome database, the specific primers were designed and used in cloning the full-length cDNA of TwKAO, the bioinformatic analysis of the sequence was performed. The qRT-PCR were used to detect the expression level of TwKAO after MeJA treatment.The full-length cDNA of the TwKAO was 1 874 bp encoding a polypeptide of 487 amino acids.The calculate molecular weight was about 56.02 kDa,and the theoretical isoelectric point (pI) was 8.89. The relative expression level of TwKAO was deduced by MeJA and reached the highest at 12 h after the treatment.Plant tissue expression analysis indicated that, TwKAO expressed the highest in leaves,while lowest in roots.For the first time, we cloned and analyzed the expression characteristics of TwKAO, which laid a foundation for deep analysis of growing development and terpenoid secondary metabolites in T. wilfordii.

Rapid screening the potential mechanism-based inhibitors of CYP3A4 from Tripterygium wilfordi based on computer approaches combined with in vitro bioassay.

CYP3A4 is the main human metabolizing enzyme, and many clinically relevant drug/herb-drug interactions (DDIs/HDIs) involving CYP3A4 are due to mechanism-based inhibition. In this study, pharmacophore model together with molecular docking (MD) are used to rapidly screen the potential CYP3A4 mechanism-based inhibitors from Tripterygium wilfordii, and in vitro experiments are conducted to validate the computational data. The results showed that the rate of computational prediction could be improved based on a combination of pharmacophore model and MD, and a combination of computational approaches might be a useful tool to identify potential mechanism-based inhibitor of CYP3A4 from herbal medicines.

Two residues determine the product profile of the class II diterpene synthases TPS14 and TPS21 of Tripterygium wilfordii.

The medicinal plant Tripterygium wilfordii (Celastraceae) contains a pair of class II diterpene synthases (diTPS) of specialized labdane-type metabolism that, despite remarkably close homology, form strikingly different products. TwTPS21 catalyzes bicyclization of the linear C20 precursor geranylgeranyl diphosphate to ent-copal-8-ol diphosphate, while TwTPS14 forms kolavenyl diphosphate. To determine the amino acid signature controlling the functional divergence of the homologues, we modeled their structures based on an existing crystal structure of the Arabidopsis ent-copalyl diphosphate synthase, archetypal of diTPSs in general metabolism of gibberellin phytohormones. Of the residues differing between TwTPS21 and TwTPS14 two located to the predicted active site, and we hypothesized that these are responsible for the functional differentiation of the enzymes. Using site-directed mutagenesis, we generated a panel of six variants, where one, or both positions were exchanged between the enzymes. In coupled heterologous assays with a corresponding class I diTPS, TwTPS2, complete product interchange was observed in variants with both reciprocal mutations, while substitutions of either residue gave mixed product profiles. Two mutants, TwTPS14:Y265H and TwTPS21:A325V, also produced ent-copalyl diphosphate, highlighting the evolutionary potential of enzymes of this family to drive rapid diversification of plant diterpene biosynthesis through neo-functionalization. Our study contributes to the understanding of structure-function relation in plant class II diTPSs and complements previous mutational studies of Arabidopsis ent-copalyl diphosphate synthase with additional examples from the specialized metabolism of T. wilfordii.

Functional characterization of NES and GES responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool in Tripterygium wilfordii.

Triptolide and celastrol, two principal bioactive compounds in Tripterygium wilfordii, are produced from geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate ((E,E)-FPP) through terpenoid biosynthesis pathway. However, little is known about T. wilfordii terpene synthases which could competitively utilize GGPP and (E,E)-FPP as substrates, producing C15 and C20 tertiary alcohols. Here we firstly cloned the genes encoding nerolidol synthase (NES) and geranyllinalool synthases (GES1, GES2), which are responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool. In vitro characterization of recombinant TwNES and TwGES1 revealed both were functional enzymes that could catalyze the conversion of (E,E)-FPP and GGPP to (E)-nerolidol and (E,E)-geranyllinalool, which were consistent with the results of yeast fermentation. Biochemical characterization revealed TwNES and TwGES1 had strong dependency for Mg2+, Km and Kcat/Km values of TwNES for (E,E)-FPP were 12.700 µM and 0.029 s-1/µM, and TwGES1 for GGPP were 2.039 µM and 0.019 s-1/µM. Real-time PCR analysis showed the expression levels of NES and GES1 increased by several fold in the suspension cells treated with alamethicin, indicating TwNES and TwGES1 are likely to utilize GGPP and (E,E)-FPP to generate tertiary alcohols as precursor of plant volatiles, which play important roles in the ecological interactions between T. wilfordii and other organisms.

The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily.

Tripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. The absence of activity-forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii.

Cloning and functional characterization of an isopentenyl diphosphate isomerase gene from Tripterygium wilfordii.

Tripterygium wilfordii Hook.F. is one of the most valuable medicinal plants because it contains a large variety of active terpenoid compounds, including triptolide, celastrol, and wilforlide. All of the pharmacologically active secondary metabolites are synthesized from the 2-C-methyl-d-erythritol 4-phosphate and mevalonate pathway in the isoprenoid biosynthetic system. The key step in this pathway is the isomerization of dimethylallyl diphosphate and isopentenyl diphosphate, which is catalyzed by isopentenyl diphosphate isomerase (IPI). In the present study, a full-length cDNA encoding IPI (designate as TwIPI, GenBank accession no.KT279355) was cloned from a suspension of cultured cells from T. wilfordii. The full-length cDNA of TwIPI was 1,564 bp and encoded a polypeptide of 288 amino acids. The bioinformatics analysis showed that the deduced TwIPI sequence contained the TNTCCSHPL and WGEHELDY motif. The transcription level of the TwIPI in the suspension cells increased almost fivefold after treatment with methyl jasmonate as an elicitor. A functional color assay in Escherichia coli indicated that TwIPI could promote the accumulation of lycopene and encoded a functional protein.

[Cloning and bioinformatics analysis of geranylgeranyl diphosphate synthase gene of Tripterygium wilfordii].

A full-length cDNA of GGPPS gene from Tripterygium wilfordii suspension cells was obtained by use of RACE strategy (GeneBank: KM978333), and then analyzed by bioinformatics approaches. TwGGPPS cDNA has 1857 nucleotides and an open reading frame (ORF) encoding a protein of 514 amino acid residues. The deduced protein has isoelectric point (pI) of 7.85, a calculated molecular weight about 57.13 kD, 5 conserved domains and 2 functional domains. PSORT Prediction showed it was located at plasma membrane. Phylogenetic analysis demonstrated that TwGGPPS1 was similar to GGPPS from other species of plants. For the first time the cloning of geranylgeranyl diphosphate synthase gene from T. wilfordii was reported, it lays the foundation for further research of diterpenoids biosynthetic pathway.

[Cloning and expression analysis of a acetyl-CoA U-acetyltransferase gene (TwAACT) from Tripterygium wilfordii].

In this study, based on the transcriptome data, we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells, and then analyzed the bioinformation of the sequence, detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934). The deduced isoelectric point (pI) was 6.10, a calculated molecular weight was about 41.20 kDa, and online prediction showed that TwAACT had two catalytic active sites. After the induction of MeJA, the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly, that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.

[Cloning and expression analysis of 4- (cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase gene in Tripterygium wilfordii].

4-(Cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase is a key enzyme in the biosynthesis pathway of terpenoids. According to the transcriptome database, the specific primers were designed and used in PCR. The bioinformatic analysis of the sequenced TwCMK gene was performed in several bioinformatics software. The Real-time fluorescence quantification polymerase chain reaction (RT-qPCR) were used to detect the expression levels of TwCMK from T. wilfordii after elicitor MeJA supplied. The results showed that the full length of TwCMK cDNA was 1 732 bp encoding 387 amino acids. The theoretical isoelectric point of the putative TwCMK protein was 5.79 and the molecular weight was about 42.85 kDa. MeJA stimulated the rising of TwCMK expression in suspension cell and signally impacted at 24 h. The research provides a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.