## 2 Results
### RNA Sequencing, Assembly, and Functional Annotation  
A total of 132.69 Gb of clean reads were obtained after processing. The clean reads obtained from each sample reached 6.5 Gb in size, with a mean GC content of 45.59% and a Q30 base percentage of 93% or above. Subsequently, all clean reads were matched and analyzed with the _Dendrobium officinal_ genome database (reference genome). Based on the comparison results, variable shear prediction, functional annotation and enrichment, gene structure optimization, and the discovery of new genes could be carried out. Ultimately, most sequences from CK (72.86-75.07%), TC7 (71.41-76.28%), TC14 (72.19-75.64%), TF (71.85-74.83%), TF7 (74.05-75.94%), and TF14 (74.17-75.11%) were matched to the _D. officinal_ reference genome. At different points in time, the numbers of reads that the assembly data mapped to the \"+\" and \"\(-\)\" chains were almost equal. According to the BLASTx analysis, 16,593, 17,669, 22,782, 22,566, 55,370, 16,803, and 22,517 assembled unigenes were annotated in the GO, KEGG, KOG, NR, Pfam, Swissprot, and Trembl databases, respectively.  
For the evaluation of protein functions, it was generally possible to search and compare assembled unigenes from the KOG protein database to classify and identify possible functions. The results showed that a total of 15,192 unigenes were annotated and divided into 25 functional categories (Figure 3). Among these categories, the number of \"general  
\begin{table}
\begin{tabular}{c c c c c c c} \hline
**Group** & **Down a** & **Up a** & **Total a** & **Group** & **Down b** & **Up b** & **Total b** \\ \hline CK\_vs\_DC7 & 60 & 81 & 141 & CK\_vs\_TC7 & 233 & 164 & 397 \\ CK\_vs\_DC14 & 68 & 49 & 117 & CK\_vs\_TC14 & 313 & 540 & 853 \\ CK\_vs\_DF & 64 & 77 & 141 & CK\_vs\_TF & 288 & 416 & 704 \\ CK\_vs\_DF7 & 68 & 97 & 165 & CK\_vs\_TF7 & 76 & 116 & 192 \\ CK\_vs\_DF14 & 82 & 52 & 134 & CK\_vs\_TF14 & 624 & 722 & 1346 \\ DC7\_vs\_DC14 & 82 & 28 & 110 & TC7\_vs\_TC14 & 508 & 854 & 1362 \\ DC7\_vs\_DF7 & 38 & 52 & 90 & TC7\_vs\_TF7 & 33 & 96 & 129 \\ DC14\_vs\_DF14 & 49 & 51 & 100 & TC14\_vs\_TF14 & 272 & 79 & 351 \\ DF\_vs\_DF7 & 27 & 62 & 89 & TF\_vs\_TF7 & 411 & 366 & 777 \\ DF\_vs\_DF14 & 48 & 21 & 69 & TF\_vs\_TF14 & 212 & 130 & 342 \\ DF7\_vs\_DF14 & 78 & 25 & 103 & TF7\_vs\_TF14 & 405 & 484 & 889 \\ \hline \end{tabular}  
* Metabolites; b genes.  
\end{table}
Table 2: Summary of the differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) among 11 library pairs.  
function prediction only\" (R: 4277 unigenes) was the highest and accounted for 28.15% of the total.  
Figure 2: (**A**) K-means analysis of differentially accumulated metabolites (DAMs). The horizontal coordinate represents the sample name, the vertical coordinate represents the content of standardized relative metabolites, and the subclass represents the metabolites class number with the same trend. (**B**) KEGG analysis of the CK and DF14 libraries. The vertical axis shows the name of the KEGG metabolic pathway, whereas the horizontal axis shows the number of metabolites annotated to the pathway and its ratio to the total number of annotated metabolites.  
In addition, \"post-translational modification, protein turnover, chaperones\" (O: 1501 unigenes) and the \"signal transduction mechanisms\" (T: 1269 unigenes) accounted for 9.88% and 8.35% of the total, respectively. The category \"extracellular structures\" (W: 35 unigenes) accounted for 0.23% of the total, which was the lowest percentage.