## 3 Discussion  
In the biosynthesis of triterpenoids in plants, the cyclization of 2,3-oxidosqualene catalyzed by OSC represents the first committed step of triterpene diversification [27], generating more than 100 different triterpene skeletons [28]. To date, more than 80 OSCs have been identified and functionally characterized in plants, mainly via the heterologous expression of cDNAs in appropriate yeast strains. In this study, we identified nine _McOSC_ genes in bitter gourd, including four _OSCs_ whose functions have been re  
Figure 4: Widetarget metabolic data assessment and differentially accumulated metabolites (DAMs) analysis. (**a**) Pearson’s correlation coefficients among the _McOSC7-OE_ samples and quality control sample (_GFP_); (**b**) The quality control PCA score chart. The same color represents three biological replicates of the same sample; the x-axis represents the first principal component and the y-axis represents the second principal component; (**c**) Volcano plot showing the differential metabolites in _GFP_ vs. _McOSC7-OE_, VIP means variable importance in project; (**d**) Fold change of camalduncic acid, enoxolone, and quinovic acid in _McOSC7-OE_ transgenic hairy roots, data represent the mean \(\pm\) SD of three independent experiments. ported previously [24], and five new ones (_McOSC3_, _McOSC5_, _McOSC6_, _McOSC7_, and _McOSC8_) (Table S2). The results of sequence alignment analyses suggested that _McOSC3_ and _McOSC6_ encode LAS and LUP, respectively, while _McOSC5_, _McOSC7_, and _McOSC8_ encode \(\beta\)-AS-like enzymes with similar catalytic functions. Gene structure, to some extent, is closely related to the function of the encoded product. In the phylogenetic tree analysis, _McOSCs_ were mainly divided into two branches (Figure 1): one branch contained genes encoding \(\beta\)-AS, LUP, and IMS-type enzymes generating the most common pentacyclic triterpenoid skeletons via a C-C-C conformation dammarenyl cation path and the other contained genes encoding LAS, CAS, and CS-type enzymes forming tetracyclic skeletons via a C-B-C conformation prosteryl cation path. This explains the significant similarity between the structure of cucurbitacins and steroidogenic triterpenes. Cucurbitacins, which are widely distributed in Cucurbitaceae plants, have a variety of pharmacological activities such as anticancer and anti-inflammatory activity [29]. Therefore, it is of great value to explore the mechanisms of cucurbitadienol formation. One strategy to increase the accumulation of active cucurbitacins in bitter gourd is to functionally modify OSCs by altering the amino acid residues involved in triterpene product specificity [30]. The catalytic activity of OSCs is mainly controlled by a few key amino acids located at its active center. Therefore, to improve the characteristics of the enzyme, it is important to understand the relationships among the position of amino acids, the structure and function of OSCs, and their catalytic mechanism. Several studies have shown that OSC family genes show distinct expression patterns in different tissues of bitter gourd and that their encoded products play diverse biological roles in resistance to pests and diseases and in adaptation to the ecological environment [31]. A previous study found that the expression level of _McCBS_ was highest in leaves and lowest in fruits [32], which was not expected. Thus, the gene transcript level may not correspond to the final levels of the reaction product in some tissues because of transport of triterpen among tissues. In the present study, we detected higher transcript levels of _McOSCI_ (encoding CS) in stems than in leaves. This may have been because we collected fruit at a different stage. If the transcript levels of _McOSCs_ change dynamically during development, it should be possible to select an appropriate collection time to maximize the triterpen content and control the degree of bitternes.