## 4 Discussion  
_R. glutinosa_ has been extensively utilized as a medicinal plant to clear away _heat_ and promote _salivation_[1]. Catalpol, the main active constituent in _R. glutinosa_, has been reported to possess anti-inflammatory, anti-oxidant, anti-diabetic, and neuroprotective effects. Previous studies have investigated catalpol accumulations, and found that different cultivars of _R. glutinosa_, as well as distinct plant samples from the same cultivar, showed diverse catalpol production abilities [41]. [PERSON] discovered a correlation between catalpol concentration and growth stage [42]. Furthermore, our unpublished data indicated that the difference in the accumulation of catalpol existed among different leaves from the same plant. Therefore, it is necessary to analyze the catalpol content before constructing _R. glutinosa_ transcriptomes. Herein, we employed HPLC to detect the content of catalpol in different cultivars and tissues. Under the condition of this study, young leaves of the Jin No. 9 cultivar exhibited a higher catalpol accumulation than that of the Beijing No. 3 and Huaifeng cultivars (Figure 2). The HPLC analysis of the different tissues of the Jin No. 9 cultivar revealed that the concentration of catalpol was significantly higher in the young leaves compared with the other parts, suggesting the young leaves were more suitable for isolating catalpol. This result was consistent with [PERSON]'s study [42]. Transcriptome analysis has been a universal tool to investigate molecular mechanisms. In the case of _R. glutinosa_, [PERSON] and [PERSON] employed transcriptome sequencing technology to identify miRNAs and genes associated with replanting disease and root development, respectively [43; 44]. RNA sequencing technology was also applied to discover functional genes involved in natural product formation in _R. glutinosa_. To explore the biosynthesis of acetoside in _R. glutinosa_, [PERSON] and [PERSON] established transcriptomic databases from the tuberous roots and the hairy roots, respectively [45; 46]. [PERSON] constructed an EST dataset from the roots and subsequently screened a number of genes in the MVA and MEP pathways, as well as GES, G10H, and 10 HGO in the upstream pathway of iridoid biosynthesis [47]. In  
Figure 10: GC-MS analysis of the products in the in vitro assays using the purified RgGES proteins (**A**) and mass spectra of the peaks from A (**B**). The protein extracted from the _E. coli_ cells harboring the empty vector pET28a was used for the control reaction. Total ion chromatograms were shown for the reaction with RgGES1 and RgGES2 yielding geraniol (peaks 1 and 2). addition, [PERSON] constructed transcriptome libraries from the radial striation and non-radial striation of tuberous roots to identify several enzymes in the downstream pathway of catalpol production, such as iridoid synthase, cytochrome P450 monooxygenase, aldehyde dehydrogenase, flavanone 3-dioxygenase, uroporphyrinogen decarboxylase, and squalene monooxygenase [27]. [PERSON] reported genome information of _R. glutinosa_ using nanopore technology. Besides the enzymes mentioned in [PERSON]'s research, [PERSON] also identified DL7H, DLGT, LAMT, and SLS in _route I_ of iridoid biosynthesis [28]. [PERSON] constructed the metabolome and transcriptome from _R. glutinosa_ dedifferentiated cells and cambial meristematic cells, and further identified inconthol dehydrogenase, cytochrome P450 family 76 subfamily A, 7-deoxyliganetin glucosyltransferase, and 8-carboxylinaolol synthase in the downstream steps of monoterenoid biosynthesis [48]. Although previous studies have identified some enzyme-coding genes in catalpol formation, there are still a number of important enzymes that have never been reported in _R. glutinosa_, such as CYP450, oxidoreductase, dehydratase, hydroxylase, and epoxidase. These enzymes probably participate in modifying the _epi_-nepetalactol skeleton to catalpol. Given the fact that catalpol is highly accumulated in the young leaves, we constructed transcriptomes from the young leaves of three _R.