## 3 Discussion  
Although _C. chinense_ exhibits important pharmacological activities owing to its triterpenaponid saponins, biosynthesis of triterpenaponids has not been characterized. Our study aimed to identify the candidate genes that encode key enzymes related to triterpenaponid saponin biosynthesis and other secondary metabolic pathways. In this study, the transcriptomes of _C. chinense_ derived from three tissues were acquired using the BGISEQ-500 technique, resulting in 30.51 Gb of clean reads that were then assembled into 135,968 unigenes with an average length of 1195 bp. Among these unigenes, 102,154 (75.13%) were mapped to seven public databases. Compared with other medicinal plant transcriptome databases, the average length and N50 values of unigenes in _C. chinense_ (average length = 1195 bp; N50 = 1890 bp) were longer than those in _Artemisia argyi_ (average length = 926 bp; N50 = 1456 bp) [17], _Oroxylum indicum_ (average length = 1080 bp; N50 = 1783 bp) [21], and _Asarum heterotropoides_ (average length = 611 bp; N50 = 507.36 bp) [22]; these results demonstrated that our transcriptome database was of high quality. In particular, the sequence size distribution was homogeneous and 10,942 (48.61%) unigenes were longer than 1000 bp, indicating successful generation of transcriptional data. Based on GO term enrichment, we focused mainly on the categories of biological process and molecular function. The most enriched ontologies were photosynthesis (leaf versus root) and drug catabolic process (stem versus root) in biological processes, while the abundant subcategory of molecular function was oxidoreductase activity (leaf versus root and stem versus root), which might be significant for the activity of cytochrome oxidase P450 in the triterpenaponid saponin biosynthesis. Figure 9: qRT-PCR analysis of unigenes encoding enzymes involved in triterpenaponid biosynthesis. Relative expression of **(A)** CL2016.Contig3 (DXS), **(B)** CL10703.Contig.3 (DXR), **(C)** CL10042.Contig3 (HDS), **(D)** CL8457.Contig10 (PMK), **(E)** CL10625.Contig5 (IDI), and **(F)** CL7015.Contig4 (FPPS) was analyzed by qRT-PCR using the actin gene (Unigene1915) as the reference gene for normalization. Red lines indicate the FPKM values of these unigenes, and blue lines indicate the qRT-PCR results. KEGG pathway enrichment analysis led to the identification of 708 unigenes relevant to triterpen saponin biosynthesis. Among these, we found that unigenes encoding DXS (CL2016. Contig 1, 3, 5, 7, and 8), and DXR (CL10703. Contig 1, 2, 3, and 6) were more highly expressed in leaves compared with other tissues. Studies showed that DXS and DXR were the key enzymes in the MEP pathway [23; 24]. In previous studies, overexpression of DXS gene in kiwifruit directly resulted in a significant increase in monoterenoid levels in transgenic tobacco leaves [25], and overexpression of DXR in _Artemisia annua_ L led to an approximately 2-fold increase in artemisinin production by greatly influencing the biosynthesis of terpenoids [26]. The expression levels of unigenes encoding DXS (CL2016. Contig 3), DXR (CL10703. Contig 3), HDS (CL10042.Contig3), PMK (CL8457.Contig10), IDI (CL10625.Contig5) and FPPS (CL7015.Contig4) were determined by qRT-PCR to verify our transcriptional data were authentic and reliable. Characterization of these unigenes contributed to our understanding of the molecular mechanisms underlying triterpen saponin biosynthesis. Studies have shown that triterpen saponin possess two conformations (\"chair-chair-chair\" and \"chair-boat-chair\"), which form the precursors of steroids and triterpenoids cyclized by 2,3-oxidosqualene cyclases [12]. The main active ingredients of _C. chinense_ are oleanane-type triterpen saponins [3]. \(\beta\)-AS is thought to catalyze the cyclization of 2, 3-oxidosqualene to form \(\beta\)-amyrin, the basic triterpene backbone of oleanane-type saponins [11]. This step is a critical branching point for phytosterol and triterpenoid biosynthesis [27; 28]. Six \(\beta\)-AS unigenes were identified in _C.