## 2 Results  
### Identification of OSC Gene Family in Bitter Gourd  
To identify members of the _OSC_ gene family in bitter gourd, BLAST searches were performed at the bitter gourd genome database. The search queries were related protein sequences from multiple species, including _Oryza sativa L., Panax notoginseng, P. ginseng_, and _A. thaliana_ (the corresponding NCBI registration numbers are LOC_Os02g04690, LOC_Os02g0470, LOC_Os02g04730, LOC_Os02g04760, LOC_Os06g28820, LOC_Os11g08569, LOC_Os11g18366, LOC_Os11g35710,BAD34645, AJR21209, AJR21210, NP_001292630, AEM42982, ABB76766, NP_001310684, XP_015579761, XP_01581645, XP_015581646, BAA33461, BAA33722, AGG09939, and AIE17466). The sequences of 11 functional _A. thaliana_ OSC proteins were downloaded from the TAIR website ([[https://www.arabidopsis.org/](https://www.arabidopsis.org/)]([https://www.arabidopsis.org/](https://www.arabidopsis.org/)), accessed on 1 June 2020). In this way, highly similar bitter gourd protein sequences were screened as candidate OSCs and were renamed sequentially as McOSC1, McOSC2, McOSC3, McOSC4, McOSC5, McOSC6, McOSC7, McOSC8, McOSC9, McOSC10, McOSC11, and McOSC12. The 12 genes encoding these proteins were further identified based on the annotation results in NCBI. _McOSC11_, _McOSC12_, and _McOSC2_ were found to encode the same sequence. _McOSC10_ and _McOSC3_ were alternative splicing variants. For those sequences identified as alternative splicing variants, the longest one was selected for subsequent analysis. Finally, nine McOSC proteins were identified, comprising 611 (McOSC5) to 768 (McOSC9) amino acids, with theoretical isoelectric points (pls) ranging from 4.90 (McOSC9) to 4.98 (McOSC5) (Table S2).  
To explore the evolution of McOSC proteins, their amino acid sequences were compared with a focus on the two highly conserved sequences: the DCT(A/S)E motif and the QXXXXXW motif (Figure S2). Studies have shown that the highly conserved DCTAE sequence is the substrate binding activity center [25; 26]. In McOSC1, McOSC2, McOSC5, and McOSC8, an Ile residue was located two residues upstream from the DCTAE motif, whereas a Val residue was located at that position in McOSC3, McOSC4, McOSC6, and McOSC9. Notably, in contrast with the findings of previous studies, an LICYCR motif was detected in McOSC5, McOSC6, McOSC7, and McOSC8. The Leu of the MLCYCR/ILCYSR sequence is conserved in lupel synthas (LUPs). According to annotation information from the NCBI, only McOSC6 is a LUP, and the other three proteins are \(\beta\)-AS-like proteins. The differences in these sequences result in the production of different triterpene metabolites. The alignment analysis revealed three highly conserved repetitive QXXXXXW motifs, which may play an important role in stabilization during catalysis in oxidosqualene cyclases and squalene cyclases across various species. The MWCXCR motif and WVEDPN motif were only present in certain McOSC proteins [24], suggesting that functional differentiation has occurred among this gene family. The analyses also revealed that the amino acid residue at the X site of the MWCXCR motif differs in lanosterol synthase (LAS) (McOSC3), cucurbitadienol synthase (CS) (McOSC1), and cycloartenol synthase (CAS) (McOSC2), while Tyr is present at the X site of \(\beta\)-AS (McOSC4) and isomultiflorenl synthase (IMS) (McOSC9).