## 2 Results and Discussion
### Results
#### 2.1.7 Identification of Candidate Genes Involved in Ginsenoside Biosynthesis
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Ginsenoside** & **Name of** & **Number of** & **Up-Regulated** & **Down-Regulated** & **Not DEGs** \\ \hline \multirow{6}{*}{Step I} & AACT & 3 & 1 & 2 & - \\  & HMGS & 9 & 2 & 7 & - \\  & HMGR & 15 & 4 & 11 & - \\  & MVK & 20 & 14 & 6 & - \\  & PMK & 16 & 10 & 6 & - \\  & MDD & 2 & 1 & 1 & - \\  & IDI & 2 & 1 & 1 & - \\ \hline \multirow{6}{*}{Step II} & GPS & 24 & 8 & 16 & - \\  & FPS & 1 & 1 & 0 & - \\  & GGPPS & 14 & 6 & 8 & - \\  & SS & 10 & 10 & 0 & - \\  & SE & 11 & 7 & 4 & - \\ \hline \multirow{6}{*}{Step III} & DDS & 1 & 1 & 0 & - \\  & \(\beta\)-AS & 9 & 2 & 7 & - \\  & CAS & 17 & 1 & 16 & - \\ \cline{1-1}  & LS & 2 & 0 & 2 & - \\ \cline{1-1}  & P450 & 335 & 161 & 168 & 6 \\ \cline{1-1}  & GT & 142 & 48 & 92 & 2 \\ \cline{1-1}  & UGT & 116 & 71 & 45 & - \\ \hline \hline \end{tabular}  
* AACT, acetyl-CoA acetyltransferase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MDD, mevalonate-5-diphosphate decarboxylase; IDI, isopentenyl diphosphate isomerase; GPS, geranyl diphosphate synthase; FPS, farnesyl diphosphate synthase; GGPPS, geranyldigmosphate synthase; SS, squalene synthase; SE, squalene epoxidase; DDS, dammareenediol synthase; \(\beta\)-AS, beta-amyrin synthase; CAS, cycloartenol synthase; LS, lanosterol synthase; P450, cytochrome P450; GT, glycostransferase; UGT, UDP-glycostransferase; DEGs, differentially expressed genes.  
\end{table} 
Table 2: Differentially expressed unigenes encoding enzymes in the ginseng biosynthesis pathway. of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) that are the universal precursors for isoprenoids biosynthesis including ginseng in all organisms [36]. So far, one _IDI_ candidate from the 454-EST dataset of 4-year-old untreated _P. ginseng_ root was identified [15]. However, no _IDI_ was discovered from MeJA-treated hairy roots of 4-year-old _P. ginseng_ using traditional Sanger EST analyses [10]. But two unigenes encoding IDI were found from our dataset that may indicate the high coverage of Illumina/Solexa sequencing platform. **Step II**: We identified several candidate genes encoding geranyl diphosphate synthase (GPS), farnesyl diphosphate synthase (FPS), squalene synthase (SS) and squalene epoxidase (SE) involved from IPP to 2,3-oxidosqualene. In a previous study, two unigenes for GPS in 4-year-old _P. ginseng_ root were detected [15], while no _GPS_ gene for this enzyme was found in 11-year-old _P. ginseng_[34]. Here, 24 _GPS_ unigenes were discovered including eight up-regulated and 16 down-regulated candidates. In a study, the expression of the _FPS_ was shown to be up-regulated by MeJA in _P. ginseng_ hairy roots [37]. We found one _FPS_ unigene from our dataset that was also up-regulated. FPP is converted to squalene under the action of SS. This is the first enzymatic reaction from the central isoprenoid pathway  
Figure 7: Putative ginseng biosynthesis pathway in _P. ginseng_. Enzymes encoded by unigenes found in this study are circled. MVA, mevalonate; MVAP, mevalonate phosphate; MVAPP, mevalonate diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate. towards sterol and triterpene biosynthesis [11] and may be a potential point for their biosynthesis regulation. Recent studies showed that only one unigene encoding SS was found in both 4- and 11-year old untreated _P. ginseng_ root [15,34]. In another case, two up-regulated _SS_ candidates were discovered from MeJA-treated hairy roots based on Sanger EST analyses [10]. It is noteworthy that 10 _SS_ candidates (Table S6) encoding SS enzymes for squalene synthesis were found in this study and all of them were up-regulated, indicating that squalene production was very active in our 4-year MeJA-treated _P. ginseng_ adventitious roots. Phylogenetic analysis revealed that some _SS_ genes closely formed one branch in the tree, even though they were derived from different plant species (Figure 8).