## 2 Results and Discussion
### Gene Function Annotation and Classification
Following the formation of the acyclic terpenoid structural building blocks, terpene synthases act to generate the main terpene carbon skeleton, and the cytochrome P450 (CYP450) superfamily may catalyze these reactions [42]. However, CYP450 enzymes form one of the largest gene families, with over 127 plant cytochrome P450-families being described [43]. The number of CYP450s involved in genitopicoside biosynthesis remains unclear. Most terpenoid-related CYP450s are members of the CYP71 clade, a large group that comprises CYP450s involved in the metabolism of specialized compounds [44]. In the _G. rigescens_ transcriptome data, 169 putative CYP450s transcripts were identified that belong to 60 families as dictated by the standard CYP family categories (Table S5), and the majority are CYP716B2 family members (20 unigenes). In the differential expression analysis, several _CYP450_ genes were screened out. Some which had Open Reading Frames (ORFs) with a BLASTX score of _E_-value \(<\)10\({}^{-5}\), were then verified by RT-PCR and sequencing. Phylogenetic analysis of the deduced protein sequences with P450s from _Arabidopsis thaliana_ revealed that five of them (GrCYP4, GrCYP5, GrCYP11, GrCYP16, and GrCYP17) belong to the CYP71 clan (Figure 9). qRT-PCR results showed that the selected genes _GrCYP4_, _GrCYP8_, and _GrCYP15_ were highly expressed in leaves, however, _GrCYP10_ and _GrCYP16_ genes were more abundant in roots (Figure 8). Figure 8: The expression pattern of three selected MEP pathway genes and _CYP_ genes in roots and leaves in _G. rigescens_. Means \(\pm\) SE; each qRT-PCR was biologically repeated three times. In the secoiridoid biosynthesis pathway, IPPs and DMAPPs are condensed into GPP by GPPS, which is then converted to geraniol by GES. Geraniol is catalyzed to 8-oxogeraniol by geraniol 8-oxidase (G8O, also named G10H) [42,45], and then to 8-oxogeraniol by 8-hydroxygeraniol oxidoreductase (8 HGO, also called G10H) [42,45]. Figure 9: Phylogenetic analysis of CYP450s from _G. rigescens._ Amino acid sequences were aligned using the CLUSTALX2 program, and evolutionary distances were calculated using MEGA6 software with the Neighbor-Joining statistical method and Poisson model. The bootstrap replications were set to 1000. The GenBank accession numbers of the sequences are GrCYP1 (KP218047), GrCYP2-1 (KP218048), GrCYP2-2 (KP218049), GrCYP3 (KP218050), GrCYP4 (KP218051), GrCYP5 (KP325125), GrCYP6 (KP218052), GrCYP8 (KP325126), GrCYP9 (KP218053), GrCYP450-10 (KJ829649), GrCYP11 (KP218054), GrCYP13-1 (KP218055), GrCYP13-2 (KP218056), GrCYP15 (KJ829650), GrCYP16-1 (KP218057), GrCYP16-2 (KP218058), and GrCYP17 (KF941188). The sequences of _Arabidopsis thaliana_ come from TAIR (Available online: [[https://www.arabidopsis.org](https://www.arabidopsis.org)]([https://www.arabidopsis.org](https://www.arabidopsis.org))). named 10 HGO) [42,45]; 8-oxogerani is sequentially catalyzed into loganin via several steps including iridoid synthase (IS), IO, 7-deoxyloganetic acid glucosyltransferase (7-DLGT), 7-deoxyloganic acid hydroxylase (DL7H), loganic acid _O_-methyltransferase (LAMT), and secologanin synthase (SLS) [45, 46, 47]. In _Catharanthus roseus_, G10H, SLS, and DL7H were three important enzymes of the monoterenoid biosynthesis pathway [48, 49, 50]. In the _G. rigescens_ transcriptome, there were annotated two _GrGPPSS_s, one _GrGES_, 18 _GrG10H_s, six _Gr8 HGO_s, 24 _GrSLS_s, one _Gr7 DLH_, one _GrIO_, one _GrIS_, and one _Gr7-DLGT_, but no sequence annotated as _GrLAMT_. Of interest was that _GrIO_, _GrIS_, _Gr7-DLGT_, and _GrCYP1_ were only expressed in leaves. Differential expression analysis identified five genes _GrGPPS1_, _GrGES_, _GrG10H_, _Gr7 DLH_, and _GrCYP1_, which were upregulated 10 times more in leaves than in roots (Table 5). Meanwhile, there were three _Gr8 HGO_s, one _GrSLS_, one _GrIS_, one _Gr7-DLGT_, one _GrCYP3_, and one GrCYP7, whose expression was five times higher in leaves than in roots (Table 5).