# Cloning and Characterization of Two Iridoid Synthase Homologs from _Swertia Mussotii_
## 3 Discussion  
_S. mussotii_ plants grow mainly on the Qinghai Tibetan Plateau at altitudes greater than 3800 m, and their seeds germinate poorly when planted at low elevations [1]. Given that the entire plant is used in Tibetan medicine, overexploitation of this plant has resulted in limited wild resources [1]. Therefore, seeking other resources, e.g., growing the plant in other areas and/or developing organ or tissue culture systems, could represent a good strategy to address the gradual shortage in the _S. mussotii_ plant supply. However, secondary metabolic pathways often change, and the reactive compounds in the cells or tissues of these systems accumulate much less compared to naturally growing plants. To regulate the production and accumulation of reactive compounds in the plant tissues, we should elucidate the related metabolic pathways, characterize genes involved in the metabolism and reveal their expression regulations. Similar secondary metabolism pathways have been demonstrated in different plants, especially for the early biosynthetic steps. _C. roseus_ has been used to characterize the secoiridoid biosynthesis pathway, and the gene encoding a key reaction step from 8-oxogerani to iridoids, namely iridoid synthase (IS), has been recently identified [13]. IS genes belong to the P5\(\beta\)R gene family, and almost all P5\(\beta\)R proteins can reduce several substrates, such as progesterone, 8-oxogerani and 2-cyclohexen-1-one, using NADPH as a co-substrate [15]. The homologous genes have also been studied in other species, underscoring their importance in secoiridoid biosynthesis [17; 18]. In this study, based on the results of sequence and transcriptomic data mining in _S. mussotii_, we proposed the secoiridoid biosynthesis pathway (Figure 1). We also recognized three P5\(\beta\)R gene homologs and characterized two of these genes (SmIS1 and SmIS2). For the third homolog, we only obtained a partial mRNA sequence of 1008 nt, which exhibits 60.42% similarity to the corresponding portion of SmIS1 and SmIS2. As the 5' end sequence is missing, we will clone it using 5' RACE in the future. Sequence and phylogenetic analyses demonstrated that the two SmIS genes belonged to the P5\(\beta\)Rs group, the members of which convert progesterone to 5\(\beta\)-pregnane-3,20-dione in cardenolide-producing plants. This finding is in accordance with the statement that all IS proteins studied to date in cardenolide-free plants exhibit high sequence similarity with P5\(\beta\)Rs [17]. The 3D model demonstrated strong conservation of the secondary structural elements between the modelled SmIS1 and SmIS2 proteins and CrIS and that the entire structure of IS is composed of a typical dinucleotide binding \"Rossmann\" fold (a characteristic feature of the SDR superfamily) [19; 20] covered by a helical C-terminal extension (Figure 3). These results suggest that ISs are orthologues that are derived from a common ancestor in the P5\(\beta\)R family, which is similar to the findings reported \begin{table}
\begin{tabular}{l l l l l l l} \hline \hline
**Enzyme** & \multicolumn{4}{c}{**Subcellular Localization Prediction Methods**} \\ \hline  & \multicolumn{2}{c}{**WOLF PSORT**} & \multicolumn{2}{c}{**Predotar**} & \multicolumn{2}{c}{**ProtComp**} \\ \hline  & ER & 6 & Mito & 0.09 & Cyto & 8.44 \\ SmIS1 & Chlo & 5 & Plastid & 0.01 & ER & 0.13 \\  & Mito & 1 & ER & 0 & Chlo & 0.84 \\  & Plastid & 1 & Else & 0.9 & Vacuolar & 0.38 \\ \hline  & Cyto & 9 & Mito & 0.01 & Cyto & 8.32 \\ SmIS2 & Cysk & 3 & Plastid & 0.01 & ER & 0.27 \\  & Plastid & 1 & ER & 0 & Chlo & 0.82 \\  & & Else & 0.98 & Vacuolar & 0.43 \\ \hline \hline \end{tabular} For WOLF PSORT, Predotar and ProtComp predictions, the favourable localizations are reported with corresponding scores. ER, endoplasmic reticulum; Chlo, chloroplast; Mito, mitochondria; Cyto, cytoplasm; Cysk, cytoskeleton.  
\end{table} 
Table 2: Prediction of SmIS1 and SmIS2 subcellular localization using website programs. in olive [17].