## 4 Discussion
### Gene Functions  
The _HMGS_ gene is involved in an important step of the MVA pathway and is an important gene for the synthesis of sesquiterpenes and triterpenes. It has been cloned from many plant species, including _Arabidopsis thaliana_[14], _Triptergium wilfordii_[39], and _Camptotheca acuminata_[16]. Therefore, _HMGS_ has received increased attention and has been extensively researched in angiosperms [16; 19]. However, in-depth research on the role and specific functions of this gene and its biosynthesis in gymnosperms has not been performed. Two amino acids encoded by the _GbHMGS_ genes obtained by cloning in this study have typical HMGS enzyme action sites and structural domains, which are indispensable constituent sites for the primary function of HMGS. Proteins are products of gene expression that play a role in life activities and constitute direct manifestations of biological function [40]. Proteins are located in different parts of the cell and have different functions. The localization of a protein plays a crucial role in the clarification of its functions and its related metabolic pathways. Therefore, knowledge of the subcellular localization of a protein is necessary for understanding its functions. Subcellular localization and Western blot analyses and the use of fluorescence (GFP)-tagged proteins have indicated that BiJHMS1 is a cytoplasmic protein [41], whereas other plant HMGSs have been predicted to be localized only in the cytosol. Through subcellular localization assays, the present study showed that GbHMGS1 is localized in the cytoplasm. The cytoplasm is the main component of metabolism, and most chemical reactions occur in the cytoplasm, which also regulates the nucleus [38; 42]. Thus, GbHMGS1 might be involved in important metabolic events and can also provide the substrates required for the function of some organelles.  
Using prokaryotic expression and functional complementation assays, [PERSON] et al. [19] performed a preliminary functional study of the _GbHMGS1_ gene. However, because plants are eukaryotic organisms, transferring the _GbHMGS1_ gene into cells of a eukaryotic plant species (_Populus_) provides more convincing evidence of the function of the protein encoded by the transgene. Two studies on ginkgo transformation [20; 21] have been published, but complete tissue cultures and transgenic systems for ginkgo remain unavailable. Thus, we further studied the functions of GbHMGS1 by using plants with a perfect transgenic system for exogenous overexpression. Although _Arabidopsis thaliana_ is the most thoroughly studied herbaceous plant, its biomass is too small for subsequent nontargeted GC-MS metabolomics of individual samples. In contrast, ginkgo is a woody plant that has a relatively distant relationship with herbaceous plants. _Populus_, as the model tree species of woody plants, has been thoroughly studied in the field, and a stable transgenic system has been established. Therefore, the exogenous _GbHMGS1_ gene was overexpressed in _Populus_ to further assess its function. In addition, removing the N-terminal domain to feedback-regulate the terpenoid synthesis on the pathway is another strategy that helps deeply study the key regulatory step of this pathway in the future.