## 2 Results
### Transcriptome Analysis of Active Ingredients
#### 2.6.1 Diterpene Biosynthesis  
Diterpene lactones such as andrographolide, neoadjuvant, and dehydrodrographolide are characteristic compounds in _A. paniculata_. Current research shows that DMAPP and IPP, which are precursors of terpene biosynthesis, are biosynthesized by the MVA and MEP pathways (Figure 7A) [13; 14]. The MVA pathway uses acetyl  
Figure 6: Determination of the contents of diterpene lactones. (**A**) andrographolide, (**B**) neoadjuvant, and (**C**) dehydrodrographolide. Values are expressed as means and standard deviation. Significant differences (\(p<0.05\)) were analyzed using Origin2019 and are indicated by letters a, b, and c in the leaf, stem, and root. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Pathway Name** & **KO ID** & \begin{tabular}{c} **Input** \\ **Number** \\ \end{tabular} & \begin{tabular}{c} **Leaf vs. Stem** \\ **DEGs** \\ \end{tabular} & \begin{tabular}{c} **Leaf vs. Root** \\ **DEGs** \\ \end{tabular} & \begin{tabular}{c} **Stem vs. Root** \\ **DEGs** \\ \end{tabular} \\ \hline \begin{tabular}{c} Phenylalanine metabolism \\ \end{tabular} & Ko00360 & 55 & 7 & 16 & 11 \\ Terpenoid backbone biosynthesis & Ko00900 & 93 & 5 & 20 & 20 \\ \begin{tabular}{c} Diterpene biosynthesis \\ \end{tabular} & Ko00904 & 23 & 5 & 11 & 11 \\ \begin{tabular}{c} Phenylpropanoid biosynthesis \\ \end{tabular} & Ko00940 & 114 & 17 & 48 & 37 \\ \
\begin{tabular}{c} Flavonoid biosynthesis \\ \end{tabular} & Ko00941 & 26 & 2 & 10 & 8 \\
\begin{tabular}{c} Flavone and flavonol biosynthesis \\ \end{tabular} & Ko00944 & 3 & - & - & - \\ \hline \hline \end{tabular}
\end{table} 
Table 3: KEGG pathways of diterpene lactones and flavonoids. CoA as the raw material. It generates DMAPP and IPP through the action of several enzymes, including acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutary-1 CoA synthase (HMGS), 3-hydroxy-3-methylglutary CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate pyrophosphate (MVD), and ispentenyl diphosphate isomerase (IDI). The MEP pathway utilizes pyruvate and G3P as raw materials and involves enzymes such as 1-deoxy-D-xylulose, 5-phosphate synthase (DXS), 1-deoxy-D-xyluloses-phosphateduetoisomerase (DXR), 2-c-methyl-d-erythrol 4-phosphacetyltidyltransferase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythrol kinase (CMK), 2-C-methyl-D-erythrol-2,4-cyclodextrinihosolate synthase (MCS), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), and isopentenyl monophosphate kinase (IPK). Research has demonstrated that diiterpene lactone biosynthesis primarily proceeds via the MEP pathway [27]. The precursors DMAPP and IPP condense in a 1:3 ratio by geranyl-aryl diphosphate synthase (GGPS) to form geranylgeranyl diphosphate (GGPP) [28]. GGPP is subsequently converted to _ent_-copalyl diphosphate (_ent_-CPP) by _ent_-copalyl diphosphate synthase (_ent_-CPS) [29]. However, the downstream enzymatic steps remain unclear. Based on the biosynthetic pathways elucidated in other diterpene-producing plants, such as _paclitaxel_[30], _Tripterygium wilfordii_[31], and _Salvia miltiorrhiza_[32], it is hypothesized that _ent_-CPP undergoes dephosphorylation by a kaurene synthase-like (KSL) diterpene synthase followed by successive oxidations mediated by various CYP450 enzymes to yield the diverse diterpene lactones [16; 33; 34]. According to the transcriptome analysis results, 120 genes were annotated to the above diterpene biosynthetic pathways, including 22 DEGs (Figure 7B; Table S9). A heat map was drawn based on the FPKM values of DEGs in _A. paniculata_, showing differences in the expression of diterpene lactone biosynthesis-related genes in the leaf, stem, and root. The highest expression levels were found in the leaf, followed by the stem and the root. This is consistent with our content determination results, which show that diterpene lactones  
Figure 7: Biosynthesis pathway of diterpene lactones and the heatmap analysis of their DEGs. **(A)** Predicting the biosynthetic pathway of diterpene lactones.