## 2 Results
### Widely Targeted Metabolomics Analysis of Transformed Hairy Roots of Bitter Gourd  
To establish a bitter melon system that accumulates functional triterpenoids, cotyled explants were transformed with _Agrobacterium_ harboring the _McOSC7_ sequence. The positive hairy root transformants were verified by PCR amplification of the heterologous sequence. Explants that were not infected did not grow roots in subsequent cultures, so bitter gourd roots with empty vector (_GFP_) were used as the blank control (Figure 3a,b). The results of hairy root qRT-PCR analysis confirmed that _McOSC7_ was overexpressed in  
Figure 1: Phylogenetic, exon–intron structure, and motif analysis among nine McOSCs. (**a**) Phylogenetic relationships among nine McOSC proteins; (**b**) exon–intron structure analysis among nine _McOSC_ genes; (**c**) motif analysis among nine _McOSC_ proteins. Types of OSCs are \(\beta\)-AS: \(\beta\)-amyrin synthase; LUP: lupel synthase; IMS: isomultiflorenl synthase; LAS: lanosterol synthase; CS: cucurbitadienol synthase; CAS: cycloartenol synthase.  
transformed hairy root material, with an average 2.5-fold increase in its transcript level over that in the blank control (Figure 3c).  
After confirming this significant increase in _McOSC7_ expression, we conducted a widely targeted metabolomics analysis to detect dynamic changes in metabolites in the transgenic hairy roots. All treatments were performed with three independent cultures (biological repetitions), with hairy roots harboring the empty _GFP_ vector as the control. Based on comparisons of mass spectrometry data with those in the local metabolic database, the metabolites of the samples were analyzed qualitatively and quantitatively. Figures S5 and S6 show the total ions current (TIC) diagram of mixed QC samples (i.e., the sum of the intensity of all ions in the mass spectrum at each time point) and the multi-peak diagram of MRM metabolite detection (XIC of multi-substance extraction), respectively. In these figures, different metabolites are represented by differently colored peaks.  
Figure 3: Identification of bitter gourd hairy roots and gene transcript level analysis. (**a**) Pictures of _McOSC7_ overexpression (_McOSC7-OE_), _GFP_ empty, and uninfected bitter gourd (WT) roots; (**b**) Results of PCR analyses of _Actin_ in _McOSC7-OE_, _GFP_ empty, and WT roots; (**c**) Relative gene transcript levels in _GFP_ and _McOSC7-OE_ transgenic materials.  
Figure 2: Relative transcript levels of _McOSCs_ in different tissues (root, stem, leaf, and fruit) of bitter gourd, as determined by qRT-PCR analyses. Data are means \(\pm\) SD from three independent experiments. a, b, c, and d indicate significant difference at \(p\) < 0.05 (independent samples \(t\)-test). Statistical analysis was performed using IBM SPSS Statistics 20.0.  
The repeatability among intragroup samples was verified by [PERSON]'s correlation analyses (Figure 4a). The results of principal component analysis (PCA) showed a trend of metabolome separation between groups, indicating that the metabolomics differed between the hairy roots overexpressing _McOSC7_ (_McOSC7-OE_) and those expressing only _GFP_ (Figure 4b). We identified 169 metabolites, including ten triterpenenes, in bitter gourd hairy roots (Table S3).  
Significantly differentially accumulated metabolites (DAMs) were screened according to the following criteria: fold change \(\geq\)1.5 and \(\leq\)0.5, and a variable importance projection value (VIP) of \(\geq\)1 in the orthogonal partial least squares discriminant analysis (OPLS-DA) model. Under these screening conditions, 41 DAMs were detected in _McOSC7-OE_ materials, including 27 downregulated and 14 upregulated DAMs (Figure 4c). Among them, three triterpenenoids were markedly upregulated in the hairy root materials: cucurbitacin D and enoxolone, which were present at levels 1.43 and 1.82 times higher, respectively, in the _McOSC7-OE_ materials than in _GFP_; and quinovic acid, which was present at a level 4.31 times higher in _McOSC7-OE_ than in _GFP_ (Figure 4d, Table S3).