## 2 Results and Discussion
### Candidate Genes Involved in the Biosynthesis of Triterpenoid Saponins
#### 2.2.2 Amyrin Synthaes  
As previously described, OSCs catalyse the cyclisation of 2,3-oxidosqualene to form a variety of triterpene skeletons [20], including phytosterol, dammarane, lupane and oleane (\(\beta\)-amyrin) [21]. This step is thus a critical branching point for phytosterol and triterpenoid biosynthesis. Over fifty different OSCs have been cloned from various plant species [22]. Among those OSCs, amyrin synthase catalyses the cyclisation of 2,3-oxidosqualene into \(\alpha\)-amyrin and \(\beta\)-amyrin, resulting in a chair-chair-chair-boat conformation. Nine unigenes were identified to be amyrin synthase genes in this study (see Table S4). In addition, phylogenetic analysis of these nine unigenes indicated that they exhibit close homologous relationships with \(\beta\)-amyrin synthases and multifunctional amyrin synthases (see Figure 5). This prediction was supported by the presence of \(\alpha\)-amyrin and \(\beta\)-amyrin type triterpenoids in the _I. asprella_ roots. Among the nine candidates, CL3079.Contig1 and CL481.Contig1 were found to contain a full-length cDNA, including start and stop codons and a polyA signal, using  
Figure 4: Phylogenetic tree of DXSs. This tree clearly shows that the distribution of the _I. asprella_ sequences throughout the three clades of the tree. _Aquilaria sinensis_ AsDXS3 (JX860325), _A. thaliana_ AtDXS (NM_117647), AtDXS1 (NM_113045), AtDXS3 (NM_121176), _Catharanthus roseus_ CrDXS2 (DQ848672), _Ginko biloba_ GbDXS (AY505128), _G. max_ GmDXS1 (NM_001249141), _Hevera brasiliensis_ HbDXS2 (DQ473433), _M. truncatula_ MtDXS1 (AJ430047), _Salvia militorrhiza_ SmDXS2 (FJ643618), _Vitis vinifera_ VvDXS (XM_002266889), _Zea mays_ ZmDXS1 (NM_001164333), Outgroup: _Perkinsus marinus_ PmDXS (AB284361). the online tool GENSCAN. To confirm the gene sequences, primers were designed to anneal around the predicted start and stop codons of the two genes, resulting in an expected length of approximately 2500 base pairs (bp). Both genes were successfully amplified from cDNA generated from a different _I. asprella_ root sample (see Figure 6, the sequencing result is shown in Figure S4) and will be characterised in the future. Figure 5: Phylogenetic tree of OSCs. The tree illustrates the likely gene function of 19 characterized OSC genes and nine candidate OSC unigenes found in this study. _A. thaliana_ AtOSC-multi (NM_106497), _A. thaliana_ AtOSC-ls (NM_114382), _Artemisia annua_ AaOSC-ba (EU330197), _Bruguiera gymnorhiza_ BgOSC-lu (AB289586), _C. roseus_ CrOSC-multi (JN991165), _Cucurbita pepo_ CpGSC-ls (AB116239), _G. max_ GmOSC-ba (AY095999), _G. glabra_ GgOSC-lu (AB116228), _Kandelia candel_ KoOSC-multi (AB257507), _Lotus japonicus_ LjOSC-lu (AB181245), _Luffa aegyptiaca_ LaOSC-ls (AB033335), _Malus x domestica_ MxdOSC-multi (FJ032006), _M. truncatula_ MtOSC-ba (AJ430607), _Olea europaea_ OeOSC-multi (AB291240), _P. ginseng_ PgOSC-ba (AB009030), _P. ginseng_ PgOSC-lu (AB009031), _Solanum lysopersicum_ SIOSC-multi (HQ266580), _Taraxacum officinal_ ToOSC-ls (AB025345), Outgroup: _Mus musculus_ MmOSC (NM_146006); ba is for \(\beta\)-amyrin synthase, multi is for multifunctional OSC gene, ls is for lanosterol synthase, lu is for lupeol synthase. The gene on CL3079.Contig1 (designated as _Ia4S1_) has a length of 2271 bp encoding 756 amino acids, showing 82% amino acid sequence identity to the mixed amyrin synthase CrAS from _Catharanthus roseus_. In contrast, the gene on CL481.Contig1 (designated as _Ia4S2_) is 2274 bp long encoding 757 amino acids, showing 86% amino acid sequence identity (Supplementary Figure S5) to the \(\beta\)-amyrin synthase AeAS from _Aralia elata_[23]. The QW repeat, DCTAE motif, as well as the MWCYCR motif, is present in both _Ia4S1_ and _Ia4S2_[24, 25, 26]. Multiple alignment analysis of _Ia4S1_ and _Ia4S2_ with 19 ASs randomly selected from Genbank highlights some amino acid residues conserved in mixed AS.