## 3 Results and Discussion
### Protein Domain, Conserved Motifs, and Gene Structure of GpOSC Proteins  
The proteins of the OSC family exhibit highly conserved domains and significant amino acid homology. Studies have shown that the homology among CAS enzymes can reach as high as 80% [23]. To analyze the protein domains of GpOSC proteins, a phylogenetic tree was constructed based on their protein sequences (Figure 2a). This analysis revealed a highly conserved protein domain within the GpOSC proteins, specifically identifying only one conserved domain: the PLN03012 superfamily (Figure 2b). Additionally, both conserved and unique motifs in GpOSC proteins were also identified (Figure 2c). From an overarching perspective, it was observed that motifs tended to be similar among genes located on the same branch or exhibiting close evolutionary relationships (Figure S2). Most GpOSC proteins contained between 8 and 10 motifs; however, GpOSC6 and GpOSC7 exhibited fewer motifs, with GpOSC7 containing only four motifs. It is speculated that the loss of conserved motifs may impact protein functionality. The conserved sequences DCTAE and QW (QXXXXXW) were present in motif 2, motif 9, and motif 10; notably, all eleven GpOSC protein sequences contained these specific motifs (Figure S3). A multiple sequence alignment of the eleven GpOSC proteins revealed numerous homologous sequences within these proteins (Figure S4). Previous studies have demonstrated that OSC family proteins have relatively high amino acid similarity, and their amino acid sequences generally contain a DCTAE motif along with several QW conserved motifs [17]. The D and C residues within the DCTAE motif were confirmed to play a role in initiating the OSC cyclization reaction. Meanwhile, the QW motif is crucial for stabilizing carbon cation intermediates while maintaining structural integrity during cyclization [51; 52]. Additionally, pentacyclic triterpenoid OSC proteins also contain a conserved sequence designated as MWCYCR; herein lies an important function for both W and Y residues in stabilizing the D/E ring structure. The comprehensive results indicated that although the protein sequences of most members of the _GpOSC_ gene family have undergone changes throughout the evolutionary process, the motifs remain highly conserved. These findings underscore the importance of motifs in determining the specificity of GpOSC proteins. The bioinformatics analysis of the _T. wilfordii OSC_ gene family revealed that alterations in conserved motifs at either the N-terminus or C-terminus could significantly impact the physiological function of these proteins [53]. More than 100 types of OSC products can be synthesized in plants, and previous studies have demonstrated that modifications to the amino acid sequence of OSC proteins can lead to variations in synthesized products. The  
Figure 2: Phylogenetic relationships (**a**), protein domain (**b**), conserved motifs (**c**), and gene structure (**d**) among 11 GpOSC proteins. diversity found within core sites among OSC enzymes contributes to structural variability within the _OSC_ gene family. A previous study identified that Trp259 in a \(\beta\)-AS enzyme regulates \(\beta\)-amyrin production, and mutation from this residue to Tyr261 can result in a conversion between tetracyclic and pentacyclic triterpenes [15]. The MEME and TBtools software were employed to illustrate the gene structures across _GpOSC_ members. The analysis revealed significant differences in gene structure among members of the _GpOSC_ family (Figure 2d). In terms of sequence length, _GpOSC1_ exhibited the longest sequence, while _GpOSC6_ had the shortest. Additionally, notable discrepancies were observed regarding exon and intron counts; specifically, exon numbers ranged from 18 to 36 and intron counts varied from 8 to 17. Most members that clustered on similar branches or within close evolutionary distances displayed comparable structures as well as similar sequence lengths and exon/intron numbers (e.g., _GpOSC2_ and _GpOSC3_).