Research On Transcriptome Analysis Of MeJA-induced Aquilaria SinensisRoot

Agarwood is an expensive resinous heartwood derived from Aquilaria plants, Only wounded trees can produce agarwood. All agarwood-producing plants are timber species which take a considerably long time to grow and the resinous portion is formed inside of the wood. Owing to a lack of genomic information, the molecular basis of wound-induced agarwood formation remains unknown. So it is desirable to produce agarwood through artificial to solve this problem. The major components of agarwood are sesquiterpenes and phenylethyl chromones, This research studied only sesquiterpenes and their biosynthetic genes.1. ObjectiveThe purpose of this study was to explore molecular basis of MeJA-induced sesquiterpenes biosynthesis and the difference between different root material.2. MethodsThis study selected the roots from aeroponic and tissue culture of Aquilaria sinensis as materials. After using 100μmol/L MeJA treatment of 72h (untreated roots for the control), using SPME-GC/MS detection sesquiterpenes of different root material. To obtain reference Aquilaria sinensis transcriptome for roots, six RNA-Seq libraries have been constructed using RNA from six MJ-treated root samples. We first analyzed the transcriptome data of the roots from aeroponic, focusing on looking terpene synthase gene as well as sesquiterpene biosynthesis-related signaling pathways and transcription factors. Then compared the differences between different root material.3. Results3.1 The difference between the chemical composition of different root after MeJA treatmentThe results of SPME-GC/MS showed the roots from aeroponic been treated 72h by MeJA, seven kinds of sesquiterpenes more than the control group, respectively β- -elemene, γ-muurolene, α-humulene, a-curcumene, β-farnesene, a-guaiene,8-guaiene. And the roots from tissue culture been treated 72h by MeJA, six kinds of sesquiterpenes more than the control group, respectively β-elemene, β-cedrene, a-guaiene, β-farnesene, a-humulene,β-guaiene.3.2 Transcriptome data analysis of roots from aeroponic3.2.1 The study screened out two signaling pathways:ko04020 (Calcium signaling pathway) and ko04010 (mitogen-activated protein kinase signaling pathway/MAPK signaling pathway), which may be associated with the sesquiterpenes biosynthesis. We excavated the possible eight key genes (AsCML42, AsCML37, AsCML15, AsCML11a, AsCML13, AsCML2, AsCALM, AsCML11b) on Calcium signaling pathway and possible seven key genes (AsMAPKK2, AsMAPKK6, AsMAPKK9, AsMAPK4, AsMAPK7, AsMAPKKK12, AsMAPKKK9) on MAPK signaling pathway.3.2.2 We dig out 13 transcription factors, they just upstreamed of the plant defense system, involved in at least one early signaling pathways, and had no significant correlation with terpene synthase gene. The 13 transcription factors include three WRKY transcription factors (WRKY 43, AsWRKY51b, OsWRKY23), seven EREBP/ AP2 transcription factor (AsERF21, AsERF17, AsERF113, AsERF5, AsERF107, AsERF87, AsERF106), two bZIP transcription factors (AsbZIP1, AsbZIP63) and one bHLH transcription factor (AsbHLH4).3.2.3 We excavated 41 transcription factors, of which 27 involved in at least one early signaling pathways of plants, but also significantly associated with the terpene synthase genes, including four WRKY transcription factors (AsWRKY75a, AsWRKY70, AsWRKY40, AsWRKYT), ten EREBP/AP2 transcription factor (AsRAP23a, AsERF4a, AsERF25, AsERF23, AsERF2, AsERF12, AsERF109, AsCRF1, AsAP2, AsAIL6), three bZIP transcription factors (AsbZIP61, AsbZIP43, AsbZIP4) and ten bHLH transcription factor (AsbHLH93, AsbHLH25, AsbHLH2, AsbHLH149, AsbHLH147, AsbHLH143, AsbHLH135b, AsbHLH135a, AsbHLH130, AsbHLH1). We speculated that the roots are induced by MeJA, primarily activated the corresponding WRKY, EREBP/AP2, bHLH and bZIP transcription factors by calcium signaling pathway and MAPK signaling pathways, which regulated the expression of terpene synthase gene, at the same time there is a certain overlap between the two two signal paths.3.2.4 We found 10 terpene synthase gene (alpha-humulene synthase1, germacrene-D synthase1, sesquiterpene synthase 1, sesquiterpene synthase2, sesquiterpene synthase 3, Sesquiterpene Synthase4, germacrene D synthase2, alpha-humulene synthase2, terpene synthase 4, Delta-guaiene synthase), in which alpha-humulene synthase 1 and alpha-humulene synthase 2 may produce a-humulene, Delta-guaiene synthase may produce a-guaiene and 8-guaiene.3.2.5 By using RT-PCR technology, we selected one of the most stable reference geneUBC2 from seven candidate reference genes. And tested seven target gene expression randomly, The results showed that the expression of target genes by RT-PCR validation and transcriptome sequencing were basically the same, indicating that the transcriptome data were reliable.3.3 Comparative analysis between the two materialsWe compared the changes in gene of two material after being induced by MeJA, and found that the roots from aeroponic been induced 24h or 48h, compared with the control, the KGEE enrichment pathways of differential genes are were more similar to the roots from tissue culture been induced 72h.And most differential genes in various pathways were consistent. We speculated that part of the KEGG pathways in roots from aeroponic may be more sensitive than in roots from tissue culture in response to MeJA. The terpene synthase genes were also consistent with this hypothesis.Through data analysis, it was found that although the two kinds of roots response to MeJA in different time, but the type of active terpene synthase gene mostly the same, and therefore, most of the production of sesquiterpene also consistent.4. ConclusionThe study used two materials induced by MeJA at different times, obtained a lot of chemical or genetic data. Through in-depth analysis of the 4 transcriptome data of roots from aeroponic, screened a number of genes may be associated with sesquiterpene biosynthesis, which worth as candidate genes for subsequent functional verification. This study also provides extensive transcriptome information for Aquilaria spp. and valuable clues forelucidating the mechanism of MeJA-induced agarwood sesquiterpenes biosynthesis and their regulation. By comparing the chemical test data and transcriptome data of two materials, we found that the sensitivity of different materials response to MeJA may be different, but the production mostly the same, this can provide inspiration for the choice of materials and time point of induction.