## 3 Results and Discussion
### Phylogenetic Analysis and Classification of OSC Gene Family  
To further elucidate the evolutionary relationship of the _OSC_ gene family in _G. pentaphyllum_, a phylogenetic analysis was conducted on the _GpOSC_ genes and those of other reported dicotyledonous plants, including _A. thaliana_, _P. ginseng_, and _M. channtia_ (Table S1). The _OSC_ genes from the monocotyledonous plant _Oryza sativa_ and the fungus _Ganoderma lucium_ were used as outgroups. The results indicated that all _OSC_ genes could be divided into eight distinct subfamilies (Figure 1). Upon a further analysis of various branches within the phylogenetic tree, it was observed that synthase genes encoding cycloartenol, lanosterol, and cucurbatdienol formed Group A, which included _GpOSC6_, _GpOSC7_, _GpOSC1_, and _GpOSC5_. Group B comprised gene branches responsible for encoding lupeol synthase, damnerenediol synthase, and other functions, with _GpOSC10_ and the _\(\beta\)-AS-like_ genes from _M. channtia_ clustering together, while _GpOSC4_ was identified as an _IMS_ gene. Genes encoding \(\beta\)-amyrin synthase (\(\beta\)-AS) were classified into Group C; within this group, _GpOSC11_, _GpOSC9_, _GpOSC8_, _GpOSC3_, and _GpOSC2_ formed monophyletic branches. Previous studies have suggested that cycloartenol synthase (_CAS_) and lanosterol synthase (_LAS_), both involved in sterol synthesis processes, may represent ancestral genes that evolved either directly or indirectly from _OSC_ genes [40; 46]. For instance, lower plants such as _Chlamydomonas reinhardtii_ and mosses contain only a single type of _OSC_ for sterol synthesis, whereas higher plants exhibit a broader diversity of _OSC_ types (approximately 9-16). Moreover, additional phylogenetic studies have indicated that the expansion of OSC genes in higher plants primarily arose from their ancestor _LAS_ through processes such as tandem duplication events, positive selection pressures, and divergent evolution [47], which is consistent with our findings. Interestingly, our phylogenetic results revealed that the _GpOSC_ genes did not cluster within the same clade as the _DS_ genes of species belonging to the genus _Panax_. This finding is consistent with a recent study identifying an _OSC_ gene that is co-expressed with genes involved in the triterpenothesis pathway of _G. pentaphyllum_[48]. Through the heterologous expression of a _GpOSC_ gene in brewing yeast and subsequent functional validation, \begin{table}
\begin{tabular}{l l c c c c c c c} \hline \hline \multirow{2}{*}{**Gene Name**} & \multirow{2}{*}{**Sequence ID**} & \multirow{2}{*}{**Number of Amino Acids**} & \multirow{2}{*}{**Molecular Weight**} & \multirow{2}{*}{**Theoretical pl**} & \multirow{2}{*}{**Instability Index**} & \multirow{2}{*}{**Aliphatic**} & \multirow{2}{*}{**Grand Average of Hydrophticity**} & \multirow{2}{*}{
\begin{tabular}{} \end{tabular} } \\  & & & & & & & & \\ \hline _GpOSC1_ & Gppc02G02.1 & 745.0 & 84.682.54 & 5.90 & 40.50 & 84.62 & \(-\)0.27 & Perroisomal \\ _GpOSC2_ & Gppc02G0431.1 & 760.00 & 87.741.97 & 6.75 & 47.81 & 78.68 & \(-\)0.31 & Cytoplasmic \\ _GpOSC3_ & Gppc02G061.05 & 750.00 & 86.268.80 & 8.08 & 45.64 & 81.81 & \(-\)0.26 & Cytoplasmic \\ _GpOSC4_ & Gppc02G017.1 & 741.00 & 85.668.05 & 6.23 & 49.61 & 76.36 & \(-\)0.35 & Plasma Membrane \\ _GpOSC5_ & Gppc02G1624.1 & 729.00 & 83.516.29 & 6.48 & 47.25 & 83.61 & \(-\)0.33 & Lysosomal \\ _GpOSC6_ & Gppc02G1531.3 & 356.00 & 40.133.93 & 6.46 & 42.66 & 87.33 & \(-\)0.24 & Lysosomal \\ _GpOSC7_ & Gppc02G1535.1 & 506.00 & 57.889.42 & 7.55 & 40.11 & 83.24 & \(-\)0.26 & Lysosomal \\ _GpOSC8_ & Gppc02G091.7 & 714.00 & 82.651.78 & 6.40 & 43.24 & 75.28 & \(-\)0.35 & Lysosomal \\ _GpOSC9_ & Gppc02G091.1 & 629.00 & 72.210.32 & 5.91 & 42.17 & 81.29 & \(-\)0.32 & Cytoplasmic \\ _GpOSC10_ & Gppc02G0155.1 & 749.00 & 86.355.59 & 6.61 & 47.20 & 77.86 & \(-\)0.37 & Plasma Membrane \\ _GpOSC11_ & Gppc10G190.1 & 767.00 & 88,595.63 & 6.36 & 50.30 & 75.70 & \(-\)0.37 & Cytoplasmic \\ \hline \hline \end{tabular}
\end{table}