## 2 Results
### Unigene Functional Annotation and Overview of Unigene Expression
to the \"metabolism of terpenoids and polyketides\" subcategory, and the largest numbers of unigenes were associated with terpenoid backbone biosynthesis (Figure 2A). The biosynthesis of other secondary metabolites included 14 pathways, of which the unigenes were most enriched in phenylpropanoid biosynthesis (Figure 2B). We annotated 708 unigenes involved in \"terpenoid backbone biosynthesis\" (KO00900), \"sesquiterpenoid and triterpen biosynthesis\" (KO00909) and \"steroid biosynthesis\" (KO00100) based on the KEGG database (Supplementary Figures S9-S11). Based on KEGG pathway analysis, we developed a model to summarize the biological pathways involved in triterpen saponin biosynthesis (Supplementary Figure S12). Triterpenoid saponins are synthesized by the MVA pathway in cytoplasm and mitochondria or the MEP pathway in plastids. Moreover, IPP and Dimethylallyl diphosphate (DMAPP) are the precursors of all isoprenoids, including monoter, sesquiterpenoids, triterpen saponins, steroids, and carotenoids. A total of 129 unigenes were identified as crucial for encoding seven key enzymes involved in triterpen saponin biosynthesis, including 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) (15 unigenes), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) (22 unigenes), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) (10 unigenes), squalene synthase (SS) (26 unigenes), squalene monooxygenase (SM) (18 unigenes), \(\beta\)-AS (32 unigenes), and LS (6 unigenes) (Table 2) (Figure 3). The enzymes most closely associated with triterpen saponin biosynthesis were PDMOs and GTs. Three hundred twelve cytochrome P450s and 84 GTs were annotated in this study (Supplementary Tables S2 and S3). The biosynthesis of triterpenoids was outlined based on genes encoding enzymes with FPKM > 1  
Figure 2: Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of C. chinense. (**A**) Classifications based on the metabolism of terpenoids and polyketides. (**B**) Classifications based on biosynthesis of other secondary metabolites. \begin{table}
\begin{tabular}{l l l l l l} \hline \hline
**Abbreviation** & **EC number** & **Unigene Number** & **No. in Stems** & **No. in Roots** & **No. in Leaves** \\ \hline AACT & 2.3.1.9 & 25 & 20 & 24 & 20 \\ HMGS & 2.3.3.10 & 8 & 4 & 8 & 4 \\ HMGR & 1.1.1.34 & 15 & 11 & 14 & 8 \\ MK & 2.7.1.36 & 4 & 2 & 4 & 2 \\ PMK & 2.7.4.2 & 12 & 8 & 11 & 10 \\ MDC & 4.1.1.33 & 17 & 10 & 13 & 9 \\ DXS & 2.2.1.7 & 22 & 17 & 19 & 18 \\ DXR & 1.1.1267 & 10 & 8 & 10 & 9 \\ MCT & 2.7.7.60 & 6 & 6 & 6 & 6 \\ CMK & 2.7.1.148 & 3 & 3 & 3 & 3 \\ MDS & 4.6.1.12 & 3 & 2 & 3 & 2 \\ HDS & 1.17.7.1, 1.17.7.3 & 6 & 5 & 6 & 5 \\ HDR & 1.17.7.4 & 18 & 13 & 15 & 11 \\ IDI & 5.3.3.2 & 13 & 7 & 13 & 7 \\ FPPS & 2.5.1.1, 2.5.1.10 & 55 & 42 & 53 & 37 \\ SS & 2.5.1.21 & 26 & 20 & 26 & 19 \\ SM & 1.14.14.17 & 18 & 15 & 17 & 15 \\ \(\beta\)-AS & 5.4.99.39 & 32 & 28 & 28 & 27 \\ LS & 5.4.99.41 & 6 & 4 & 5 & 4 \\ CAS & 5.4.99.8 & 20 & 14 & 16 & 13 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Number of unigenes encoding the key enzymes involved in the biosynthesis of triterpenoid saponins in C. chinese. Figure 3: Pathway for triterpenoid saponin biosynthesis in C. chinese. The expression levels of unigenes encoding key enzymes from each process are shown using a heatmap. The columns L, R, and S correspond to leaf, root, and stem samples, respectively; red and green represent high and low expression levels, respectively. As previously described, \(\beta\)-AS catalyzes the cyclization of 2,3-oxidosqualene to form triterpene skeletons, a critical branching point for phytosterol and triterpeneoid biosynthesis. Six unigenes were confirmed to encode \(\beta\)-AS in this study by aligning their amino acids to the NCBI BLAST database (Supplementary Table S4). The alignment of six \(\beta\)-AS amino acid sequences showed that their sequence identity was 81.52%, and \(\beta\)-AS contained a characteristic region (MWCYCR) and a well-conserved binding site (DCTAE) (Figure 4). We chose three unigenes (i.e., CL2196. Contig2, CL5601. Contig1 and CL17709.