## 3 Discussion  
Transcriptome sequencing is an effective tool for large-scope gene identification and expression profiling analysis. In this study, high-throughput RNA-seq was applied to characterize the transcriptomes of _M. canadensis_ treated with MeJA. Six sequencing libraries were constructed including three replicates for both CK and MeJA treatment and at least 8 Gb clean data was produced for each library. After assembly, a total of 81,843 unigenes were obtained with an average length of 724 bp. Functional annotation indicated that 64.55% of unigenes could be annotated in at least one database. This is the first reported transcriptome study of _M. canadensis_ and will provide useful information for further study of this species. Endogenous MeJA is believed to be a primary regulator of the JA signal pathway in plants. In Arabidopsis, the JA signal is perceived and transduced via the SCF\({}^{\text{COI1}}\)-JAZ co-receptor complex. In the resting state, JAZ proteins interact with and repress downstream MYC2 to suppress of JA responses. In response to JA signal, JAZs are degraded by SCF\({}^{\text{COI1}}\)-ubiquitin-proteasome, then MYC2 is released from the repressor complex to regulate gene expression [17; 31]. In this transcriptome dataset, 24 DEGs associated with JA signal transduction were identified in _M. canadensis_. Of these, 23 DEGs showed up-regulation under MeJA treatment, encoding putative JAZ (9), MYC2 (7), TPL (1), NINJA (6), and SKP1 (1). qRT-PCR was also used to verify the reliability of the RNA-Seq data. These results suggested that application of exogenous MeJA may regulate the JA signaling pathway in _M. canadensis_. Figure 7: Expression validations of 9 menthol biosynthetic genes in control and MeJA-treated samples using qRT-PCR. The RPKM values obtained from RNA-Seq data are indicated on the top of each graph. GPPS, Geranyl diphosphate synthase; LS, (\(-\))-Limonene synthase; L3 OH, (\(-\))-Limonene-3-hydroxyylase; iPD, (\(-\))-trans-Isopiperitenol dehydrogenase; iPR, (\(-\))-Isopiperitenone reductase; iPI, (\(+\))-_cis_-Isopulegone isomerase; PR, (\(+\))-Pulegone reductase; MFS, Menthofuran synthase; MR, (\(-\))-Menthol dehydrogenase. TFs are sequence specific DNA-binding proteins that interact with the regulatory regions of the target genes and regulate their expression. Several transcriptome profiling studies have indicated that MeJA treatment triggers an extensive transcriptional reprogramming of metabolism [33; 34; 35]. A lot of TFs are elicited by MeJA and participate in regulation of specific metabolic processes [36]. Here, 157 differentially expressed TFs (102 up-regulated and 55 down-regulated) belonged to 32 families were identified by annotating in PlantTFDB database. It is worth noting that the WRKY family was the largest differentially expressed family containing 21 DEGs. It is also reported that more than 25% of WRKYs are induced in response to jasmonate in _A. thaliana_ and _Catharantthus roseus_[37]. Of the 21 differentially expressed WRKYs, the number of up-regulated genes was much higher than that of down-regulated ones (19 vs. 2). Similar observations have been found in other plants. For example, 16 up-regulated and 3 down-regulated WRKY TFs were identified under MeJA treatment in _A. annua_[27]. In _Lycoris aurea_, 32 differentially expressed WRKY TFs were identified, of which 26 were up-regulated and only 6 were down-regulated [38]. The WRKY family is an important class of JA-responsive TFs that regulate plant secondary metabolism [39]. In _A. annua_, a WRKY TF _AaWRKY1_ was strongly induced by MeJA and regulated the expression of _ADS_, a key gene of artemisinin biosynthesis [21]. In _Panax quinquefolius_, a MeJA-responsive WRKY TF _PqWRKY1_ was isolated, which could positively regulate the biosynthesis of triterpene ginseng [40]. The large number of up-regulated WRKY TFs in _M. canadensis_ suggests that they may function in JA-responsive transcriptional regulation of secondary metabolism. Monoterpenes are the major constituents of _M. canadensis_ essential oils.