## 2 Results
### Sequence and Phylogenetic Analysis of Proteins Encoded by the EhFPS  
To clarify the phylogenetic relationship between the _EhFPS_ genes, the amino acid sequences of the _FPS_ genes of _E. hirta_, rubber tree, cassava, _Eucommia ulmoides_, _Tanaxacum koksaghyz_ Rodin, _Tanaxacum mongolicum_, and _A. thaliana_ were used to construct a phylogenetic tree using the neighbor-joining method (Figure 6). _EhFPS_ genes were divided into two subtribes, of which the branch of subtribe I contained all EhFPS proteins other than _EhFPS2_; _EhFPS1_ was independently separated in this branch. The other subtribe in which _EhFPS2_ was located also contained multiple proteins, such as _Taraxacum koksaghyz_ Rodin, _Tanaxacum mongolicum_, and _Eucommia ulmoides_, among which _EhFPS2_ was closely related to _HBFPS3_ in the rubber tree and _MeFPS3_ in cassava. _MeFPS1_ and _HBFPS1_, as well as _MeFPS2_ and _HBFPS2_, were independently evolved subclades. In summary, there were homologous proteins in _Rhizoma Dioscoreae_ that were closely related to the rubber tree, _Eucommia ulmoides_, and cassava, but most differentiated into independent branches. Therefore, it is speculated that it exercises a different functional mechanism from that of other plants such as rubber and cassava.  
Figure 3: The sequence motif, conserved domains, and gene structural analysis of EhFPS. **(A)** The motif prediction of gene structures. In motif pattern, different colored boxes indicate different motifs; motif1–motif10 were represented by different numbers in the boxes (1–10). **(B)** Conserved domain prediction. The conserved structure of _FPS_ genes with only one polyprene synthetase domain was predicted by NCBI-CDD, and the gray color represents the conserved structure of polyprene synthetase domain. **(C)** Gene structure map. The blue bar indicates the coding sequence (CDS), the black line indicates the intron, and the red bar indicates the untranslated region (UTR).  
Figure 4: Multiple sequence alignment of a conserved region from _E. hirta_, _H. brasiliensis_, and _A. thaliana_. Motif1 (GKXXR), motif2 (EXXXXXXXXXDXXXXXXRG), motif3 (GQXXD), motif4 (KT), and motif5 (GXXGPOXXDDXDXXXXXXXXXXXXXXXXXXXXXXXXX). Among them, the lysine residue of motif1 can promote the binding of FPPS with isopentenyl pyrophosphate (IPP). Motif2 contains the first aspartic-acid-rich region, DDXXD, which determines the length of the product chain. The amino acid residues in this region participate in the formation of the C-C bond between IPP and the allylic substrate and determine the length of the product chain. In motif3, among the three fixed conserved amino acids, G (1 st position), Q (2 nd position), and D (5 th position) are all conserved in all plant FPPS sequences. Motif4 is a conserved amino acid in all plant FPPS sequences. Motif5 contains the second aspartic-acid-rich region, DDXXD. The marked helices represent the \(\alpha\)-helices in the FPS protein, which play a role in forming the catalytic pocket or stabilizing the protein structure.  
Figure 5: Modeling of eight EhFPS proteins. The structure of eight EhFPS proteins with \(>\)90% confidence level are shown. Using the SWISS-MODEL, the EhFPS protein sequence was used for modeling. The structures of eight EhFPS proteins with \(>\)90% confidence level are shown. The finest place in the figure is the loop region, which is divided into three channels: red, green, and blue. The range of each color channel is 0–255; 0 indicates that there is no color channel, and 255 indicates the maximum intensity of the color channel. The higher the channel value, the stronger the three colors.