RNA Sequencing On Amomum Villosum Lour.-induced By MeJA Identifies The Genes Of JA Signal Transduction And Transcription Factors Involved In Terpene Biosynthesis

Amomum villosum Lour. (Fructus Amomi) is an important Chinese medicine that contains volatile terpenes, including a-pinene, β-pinene, camphene, limonene, myrcene, linalool and so on. Biosynthesis of volatile terpenoids in plants is mainly dependent on terpene synthase.The, terpene synthase in the downstream play directly impact on the volatile terpenes biosynthesis. The transcription factors might bind the terpene synthase promoters to regulate the biosynthesis of terpenes.ObjectiveAs upstream regulatory elements, transcription factors regulate both the plant hormone signal transduction and terpene biosynthesis to affect the terpenes production. Furthermore, it has great prospects to reform the transcription factors by genetically engineered to accumulate the secondary metabolites. However, the important transcription factors that ate known to be involved in terpene biosynthesis inA.villosum are relatively less well-studied.This study aimed to explore the transcription factors which involved in the terpene biosynthesis and JA signal transduction, for constructing transcription factors regulatory networks in terpene biosynthesis pathway in A.villosum. These works might filter out some important transcription factors and demonstrate their utility for enhancing the medicinal quality in A.villosum.Methods2.1 Selection of transcription factors and terpene synthases in A.villosumWe screened the differential expressed transcription factor unigenes by local blast and annotation methods in the expression profiling and transcriptome databases of A.villosum induced by MeJA. Moreover, the single longest deduced proteins for each candidate unigenes were submitted to the NCBI CDD and PlantTFcat server for conserved domain identification.With transcription factors and terpene synthase co-expression networks, we screened the key transcription factors and its co-expressed terpene synthaseunigenes. And then we screened the transcription factors and terpene synthase, might involve in terpene biosynthesis by weight correlation network analysis of transcriptome data and secondary metabolites.2.2 RNA isolation, cDNA synthesis and RT-qPCRTotal RNA was isolated from the peels and seeds, and cDNA was synthesized. Forward and reverse primers were designed according to canadiate genes and were verified, we screened the best annealing temperature for RT-qPCR. Primer pairs were tested for amplification kinetics and linearity with a standard cDNA dilution curve and new primers were designed if necessary. Expression levels were normalized using AvActin (Unigene0133538) and AvTUA (Unigene0093134) mRNA levels by 2-ΔΔCt. 2.3 Plant materia treated with high concentrantion MeJAWe selected the healthy ripe fruits and sprayed with solvent control (0.0001% tween 80) and MeJA (600 μmol·L-1 and 1000μmol·L-1 MeJA). After the spraying, the ripe fruits were packed with plastic wrap immediately. Moreover, we set blank controls and all the samples were collected 24h later.2.4 The research of endogenous JA contents in A.villosum induced by MeJAThe endogenous JA in the fresh tissues of A. villosum was extracted by BUCHI. The extraction were purified and enriched by solid phase extraction column. The eluate was concentrated and dissolved by methanol. We used LC/MS determination to detect JA content.2.5 Bioinformatics analysis of candidate transcription factorsWe predicted the subcellular localization of the protein by SubLoc and PSORT. And the localization signal peptide of transcription factors were predicted by TargetP.We blasted the canadidate genes to Arabidopsis thaliana genome in GeneBank, and search the Interact and BioGRID database to predict the canadidate TFs interactions. The best candadidate TFs DNA binding domains were predicted by JASPAR 2016 server. Finally, we use ORFfinder to analyze whether the target gene sequence contains a complete open reading frame.Results3.1 RNA sequencing on A. villosum -induced by MeJA identifies the genes of WRKY and terpene synthasesRNA sequencing on A.villosum-induced by methyl jasmonate (MeJA) revealed that thirty-six differential expressen unigenes contained complete WRKY domains. Further, we obtained six differential expressed WRKY unigenes, respectively AvWRKY28, AvWRKY6, AvWRKY72, AvWRKY21, AvWRKY75and AvWRKY40, and four related terpene synthase (TPS) unigens (i.e.,(+)-neomenthol dehydrogenase, S-(+)-linalool synthase, (+)-germacrene D synthase and myrcene synthase). Ultimately, we presumed a model by qRT-PCR datas wherein AvWRKY72, AvWRKY28 and AvWRKY40 coordinately trans-activated the AvNeoD promoter to biosynthesize monoterpene.3.2 RNA sequencing on A.villosum-induced by MeJA identifies the genes of MYC and terpene synthasesIn this study, we combined transcriptome and bioinformatics to filter out five MYC gene (respectively AvMYC4a, AvMYC4b, AvMYC4c, AvMYC2a and AvMYC2b) and five terpene synthase gene (respectively β-eucalyptol synthase, sesquiterpene synthase 3, sesquiterpene synthase A1, (+)-germacrene D synthase and linalool synthase) involved in terpene biosynthesis. Correlated RNA-Seq data with metabonomics, we screened AvMYC4a, AvMYC4b, AvMYC2b, P-eudesmol synthase (AvEud) and (+)-germacrene D synthase (AvGerD) gene involved in volitatle terpenes biosynthesis. However, AvMYC4a, AvMYC4b might not directly regulate AvEud gene. AvMYC4a might activate other terpenes synthase promoters to biosynthesize the terpenes.3.3 The effect of endogenous JA and its signal pathway genes induced by MeJA in A.villosumThe trend of endogenous JA in the peels and seeds of A.villosum were consistent. There were spatial differences in JA signal genes transcription level, which was various efficiency in response to the concentrations of MeJA. JA signal transduction genes were more in response to 200μmol·L-1 MeJA induction. There was homoousia in JA signal transduction genes expression in the plants in general, however, there was local genetic diversity betweent the plant species. According to JA signal transduction gene regulatory networks, we speculated the endogenous JA might induce AvJAR1 expression to raise AvJAZ1b protein, which interacted with AvJAZ1 a and release AvMYC2b to regulate the downstream genes expression.3.4 The candidate genes expression induced by high concentration of MeJAIn previous experiments, it might be too high solvent concentration for the solvent control to product more volatile terpenoids than the MeJA treatment And we found that there were olatile terpenoids producted induced by 600μmol·L-1 MeJA than 200μmol·L-1 MeJA suggesting that the more volatile terpenoids were producing by increasing the high concentration of MeJA. Thus, we used 0.0001% tween-80 as a solvent, and 600μmol 1000μmol·L-1 MeJA to treat with A.villosum and found that the endogenous JA concentration in the peels and seeds increased with the concertion of MeJA induced by high concertion of MeJA. The change of endogenous JA in the peels was consisted with the seeds. AvMYC2b was highly consistent with the change of endogenous JA in response to MeJA. AvMYC2b was a core element in the JA signal tranduction pathway, which indicated that the JA signal tranduction was enffected by the endogenous JA. It was predicted that AvWRKY28 and AvWRKY6 synergyly regulated AvNeoD expresstion by gene co-expression networks. AvMYC4a might interact with AvMYC4b and AvWRKY72 to transactive the AvEud promoter for the volatile terpene biosynthesis. AvMYC4a might complexes with AvMYC4b and AvWRKY72 to activatie the promoter of AvEud for regulating corresponding volatile terpene biosynthesis.3.5 The key genes involved in terpene biosynthesis networks and their bioinformatics analysisThere were spatial differences in the level of the key genes in1-deoxy-D-xylulose-5-phosphate (MEP) and Mevalonate (MVA) pathwayh, and the various transcription levels in response to different concentrations of MeJA.According to the gene regulatory networks about terpenes biosynthesis, we predicted that JA signal transduction and stimulate AvMYCs regulate the expression of terpene synthase to regualte the terpenes biosynthesis in the fruits of A.villosum was induced by MeJA. AvWRKY6, AvWRKY28, AvWRKY72 and AvWRKY40 might regulate the biosynthesis of monoterpene, and AvWRKY75 might regulate the biosynthesis of sesquiterpene. Furthermore, AvWRKY6, AvWRKY28, AvWRKY72 AvWRKY75, AvWRKY40 and AvMYC2b were putatively localized in the nucleus. Some candidate transcription factors might have interactions, except for AvWRKY28 and AvWRKY72. All candidate transcription factor hadnot complete CDS, which provided a refference for subsequent functional verification experiments.ConclusionThis paper attempted to combine metabolic and transcriptome data for obtaining five transcription factor genes(respectively AvMYC2b, AvWRKY40, AvWRKY75, AvWRKY28 and AvWRKY6) and three corresponding terpene synthase genes involved in terpene biosynthesis, which provide a target for reformming the A.villosum by genetically engineering. We combined RT-qPCR and metabolic datas construct a terpenes regulation mode tha AvWRKY6, AvWRKY28, AvWRKY72 and AvWRKY40 might mainly regulate the biosynthesis of monoterpene, and AvWRKY75 might mainly regulate the sesquiterpenes biosynthesis, which provided a clue to further research on the biosynthesis of terpene in A.villosum.