## 2 Results and Discussion
### Candidate Genes Involved in the Biosynthesis of Triterpenoid Saponins  
#### 2.2.1 Terpenoid Backbone Biosynthesis  
Terpenoids are derived from C5 isoprene units through a \"head-to-tail\" connection. The conjunction of a different number of C5 isoprene units brings about various intermediates, such as IPP (C5 unit), DMAPP (C5 unit), GPP (C10 unit), and FPP (15 unit), which form the carbon skeletons of the different terpenoids. IPP, along with its isomer, DMAPP, are important intermediates in terpenoid backbone formation; both intermediates can be synthesized through the MVA pathway in the cytoplasm, or the MEP pathway in the plastid. Genes encoding all of the essential enzymes for both pathways were found in this transcriptome data, indicating that both pathways are active in the _I. asprella_ roots. The MVA pathway is essential for the biosynthesis of sterols, sesquiterpenes and triterpenoids. Twenty-two unigenes in the _I. asprella_ transcriptome, including four AACT genes, three HMGS genes, six HMGR genes, one MK gene, five PMK genes and three MDC genes (see Table S1), were identified to be involved in the MVA pathway. Among these enzymes, HMGR catalyses the conversion of HMG-CoA into MVA, which is an irreversible, two-step biochemical reaction that reduces the thioester group into a primary alcohol [18]. Therefore, HMGR is considered an important rate-limiting enzyme in the MVA pathway. In this study, the presence of six highly homologous genes implied that HMGR might be encoded by multiple genes in _I. asprella_. Together with other plant HMGRs, these six candidate unigenes were derived from a common ancestor (see Figure 3). Monoterpenes and diterpenes are synthesised through the MEP pathway, and 23 unigenes encoding enzymes involved in this pathway, including 13 DXS genes, four DXR genes, two MDS genes and one each of MCT, CMK, HDS and HDR (see Table S2), were found in the transcriptome. DXSs catalyse the formation of 1-deoxy-D-xylulose 5-phosphate through the condensation of pyruvate and glyceraldehyde-3-phosphate. The DXS genes can be classified into three clades: the DXS1 clade is involved in primary metabolism; the DXS2 clade is responsible for secondary terpenoid biosynthesis; and the recently elucidated DXS3 clade is involved in the biosynthesis of products that are essential for plant survival but expressed at a low level [19]. Further classification was predicted for these 13 genes with other defined plant DXSs using phylogenetic analysis (see Figure 4). The results showed that four unigenes, including CL2147.contig1, CL1311.contig2 and CL2836.cotig1 and 2, were grouped into the DXS1 clade; unigene26662 was the only unigene grouped into the DXS3 clade; and the remaining eight unigenes were grouped into the DXS2 clade. \begin{table}
\begin{tabular}{c c c} \hline
**Public database** & **No. of matched unigenes** & **Annotation percentage/\%** \\ \hline Nr & 37,674 & 72.64 \\ Nt & 32,994 & 63.62 \\ Swiss-Prot & 22,661 & 43.69 \\ KEGG & 20,752 & 40.01 \\ COG & 12,860 & 24.80 \\ GO & 29,375 & 56.64 \\ Total & 39,269 & 75.71 \\ \hline \end{tabular}
\end{table} 
Table 3: Unigenes mapped to the public databases. **Figure 3.** Phylogenetic tree of HMGRs. _Arabidopsis thaliana_ AtHMGR (NM_127292), _Cucumis sativus_ CsHMGR (XM_004170850), _Cyclocarya palliurus_ CpHMGR (EU296534), _G. max_ GmHMGR (XM_003547838), _Glycyrrhiza uralensis_ GuHMGR (GQ845405), _Medicago truncatula_ MtHMGR (XM_003629008), _P. quinquefolius_ PqHMGR (FJ755158), _Populus trichocarpa_ PtHMGR (XM_002313533), _Solanum tuberosum_ StHMGR (NM_001288532), _Taxus x media_ TxmHMGR (AY277740), Outgroup: _Danio rerio_ DrHMGR (NM_001014292). Both MVA and MEP pathways produce the C5 unit IPP, which can be transformed into its isomer, DMAPP, by IDI (Isopentenyl diphosphate isomerase). Meanwhile, IPP and DMAPP are assembled into GPP, FPP and GGPP by a series of prenyl transferases, including GPPS, FPPS and GGPPS. FPP is an important intermediate of triterpen biosynthesis.