## 3 Discussion  
Triterpenoids are a class of natural products formed by condensation of isoprene as the basic unit. They are widely found in medicinal plants such as licorice, ginseng, and eleuthero and have significant anti-inflammatory, antibacterial, and antitumor properties. To date, researchers have identified six triterpenoids in _E. hirta_: 3\(\beta\)-taraxacum, taraxerone, rituximab, cycloart-23-ene-3\(\beta\),25-diol, 3\(\beta\)-9,19-cyclodextrinanost-23-ene-3,25-diol, and vomifoliol [15]. The _FPS_ gene family serves as a crucial rate-limiting enzyme in the terpene synthesis pathway in plants and significantly contributes to the production of hemipperpens and triterpenoids [16]. Its members have been identified in various medicinal plants and have been shown to play important roles in the synthesis of isoprene and triterpene saponins. The mRNA level of dammarane synthase in _Centella asiatica_ was significantly elevated in a similar study that examined the heterologous expression of the _PgFPS_ gene  
Figure 11: The subcellular localization of EhFPS-GFP fusion protein was transiently expressed in tobacco leaves. Images were captured using a confocal microscope. Control group: 35S-eGFP:35S-pCAMBIA2300-eGFP; cytoplasm marker: 35S-Os1 sSrke-RFP, 35S-_EhFPS1_-eGFP, 35-_EhFPS2_-eGFP, and 35S-_EhFPS7_-eGFP were constructed into the 35S-pCAMBIA2300-eGFP vector by homologous recombination of _EhFPS1, EhFPS2_, and _EhFPS7_, respectively. The images include the cytoplasmic marker as a red fluorescence channel (first panels) and GFP as a green fluorescence channel (second panels). The bright-field (third panel) and merged (fourth panel) images are shown on the right. Bar = 20 \(\mu\)m.  
cloned from ginseng roots in _C. asiatica_ hairy roots. This increased production of saponins and triterpenes in _C. asiatica_ results in the rapid accumulation of macdessoside [17]. _FPS_ plays a crucial role in the synthesis of bioactive compounds essential for growth and stress responses in plant species such as _Matricaria recutita_[18] and _Blumea balsamifera_[19]. Significant conservation of the _FPS_ sequence across plant families, particularly within Asteraceae, was indicated by the discovery of cDNA for _FPS_ in various species through sequencing studies. Consistently with our findings, another study reported that the full-length cDNA of the _FPS_ gene (_CnFPPS_) in _Chamaemelum nobile_ was 1239 bp long and encoded for a 342 amino acid with a molecular weight of approximately 39.38 kDa [20]. An investigation of the sesquiterpene lactone drug artemisinin revealed that overexpression of the endogenous _FPS_ gene in _Artemisia anuna_ can increase endogenous artemisinin content by 2 to 25 times [21]. The simultaneous transfer of two copies of the _FPS_ gene reduces artemisinin production in transgenic plants [22]. However, cotransformation of _A. anuna_ with the _FPS_ and _DBR2_ genes can significantly increase artemisinin content by 3.36 times [23]. Compared with the wild type, the contents of four ginseng, Rh1, Rg1, Re, and Rd, detected in the _FPS_ gene overexpression lines of _Panax notoginseng_ were significantly increased by 2.66, 1.76, 4.35, and 2.90 times, respectively [24]. However, the identification and expression analysis of _FPS_ genes in _E. hirta_ have not yet been reported. In this study, using comparative genomic methods, eight _EhFPS_ genes were successfully identified and were found to be located on chromosomes 1, 3, 6, and 8, with chromosome 6 containing five of these genes. Notably, a tandem gene cluster comprising _EhFPS4_ and _EhFPS7_ was identified. Furthermore, the results of the gene duplication analysis suggest that the evolution of these genes may have resulted in homologous gene pairs through tandem duplication and fragment duplication events, further confirming the evolutionary mechanism of _FPS_ genes in plant genomes. In addition, gene duplication analysis results suggest that the evolution of these genes may have been formed by tandem duplication and fragment duplication events.