## 2 Results and Discussion
### Results
#### 2.1.7 Identification of Candidate Genes Involved in Ginsenoside Biosynthesis
In particular, two contigs (CL4699.contig1 and CL4699.contig3) out of 10 belonging to cluster CL4699 showed close similarity to the _P. ginseng_ candidate _SS1_ up-regulated by MeJA [37] indicating that these contigs were likely to be involved in ginsenoside biosynthesis. SE converts squalene to 2,3-oxidosqualene from which phytosterol and triterpene in plants are synthesized. The number of unigenes for SE was larger in our work than that of previously identified by the 454 pyrosequencing and Sanger EST analyses in untreated and MeJA-treated _P. ginseng_ root respectively [15,34]. These results may represent the immense capacity of both MeJA and Illumina/Solexa sequencing technology. **Step III**: In _P. ginseng_, dammaenediol synthase (DDS), \(\beta\)-amyrin synthase (\(\beta\)-AS), cycloartenol synthase (CAS), lanosterol synthase (LS) and lupeol synthase (LUS) belonging to oxidosqualene cyclase (OSC) family is situated at the branching point of ginsenoside and phytosterol biosynthesis. Their biosynthesis is achieved by three reaction steps namely cyclization, hydroxylation and glycosylation [38]. In cyclization, 2,3-oxidosqualene is converted to cycloartenol by CAS and lanosterol by LS. 2,3-oxidosqualene is also converted to \(\beta\)-amyrin under the action of \(\beta\)-AS and subsequently oleanane-type ginsenoside (Ro) is synthesized. However, the absence of \(\beta\)-AS and CAS  
Figure 8: Phylogenetic analysis of 10 identified _SS_ unigenes from our dataset and other plant _SS_s. Phylogenetic tree was generated using the neighbor-joining (NJ) method in MEGA4. \(\blacktriangle\) indicates the unigenes from our current study. The box in red outline indicates one group of genes that are more similar with each other. both in 4- and 11-year old _P. ginseng_ roots [15,34] may indicate that oleanane-type ginseng (Ro) and phytosterol were not actively biosynthesized in their roots. Another branch of this step is the DDS enzyme catalyzing the production of dammarenediol-II from 2,3-oxidosqualene. The genes encoding this enzyme were shown to be up-regulated by MeJA [37,39]. DDS was shown to be absent in 11-year-old _P. ginseng_ root [34] indicating that dammarane-type ginseng biosynthesis was not active in its roots. Moreover, many genes were discovered from transcriptome analysis of 4-year-old American ginseng roots. However, no gene responsible for the cyclization of oxidosqualene was found [35]. Interestingly, suppression of CAS caused enhanced ginseng content in _P. ginseng_ hairy roots. This phenomenon was related to lower CAS and higher DDS activity, presumably due to increased availability of 2,3-oxidosqualene for DDS to produce dammarenediol-II and subsequently dammarane-type ginsengides [40]. We have identified one candidate encoding DDS induced by MeJA induction. However, out of a total 17 _CAS_ and 2 _LS_ unigenes found in our dataset, 16 candidates encoding CAS and all of the _LS_ unigenes were down-regulated indicating that lower CAS and LS activity may supply more 2,3-oxidosqualene for DDS and \(\beta\)-AS leading to the increased final ginsengide levels and reduced total phytosterol contents. However, we did not find any LUS functioning for lupeol synthesis indicating that its absence may increase the final ginsengide levels as well. In hydroxylation, two reaction steps are catalyzed by two cytochrome P450 (P450) enzymes: the conversion from dammarenediol-II to protopanaxadiol by one P450 and the conversion from protopanaxadiol to protopanaxatriol by another P450 [41,42]. One hundred and thirty-three _P450_s and 25 _P450_s candidates were identified previously in a cDNA library generated from 4-year-old MeJA-induced and 11-year-old untreated hairy roots of _P. ginseng_ respectively [10,34]. In another case, among _27 P450_s, six were up-regulated and eight were down-regulated in response to MeJA [17]. By comparison, we found a total of 335 _P450_s unigenes in our study; of those 161 were up-regulated and 168 down-regulated and six candidates did not show any differential expression level.