Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg.

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.

[Prokaryotic expression and purification of Aquilaria sinensis(Lour.) Gilg AsMYC2 protein].

The MYC2 transcription factor is a member of the important plant b HLH transcription factor families, and it is also the core regulatory elements in jasmonate（JA） signaling pathway. However, there is a little information about AsMYC2 gene in Aquilaria sinensis. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length coding sequence (CDS) of AsMYC2 gene was amplified using RT-PCR method and subcloned into pGEX-4T-1 vector by the gene recombination technique. The recombinant vector pGEX-4T-1-AsMYC2 was verified by restriction enzyme digestion and nucleotide sequencing, and was transformed into E. coli BL21（DE3） to express the protein. A maximum expression of soluble protein was observed with induction by 0.1 mmol·L(-1) IPTG at 37 ℃ for 4 hours. The fusion protein was purified through a Sepharose-Glutathione column, and verified by SDS-PAGE and Western blotting using an anti-GST polyclonal antibody. We successfully constructed the GST-AsMYC2 plasmid, produced and purified the GST-AsMYC2 fusion protein, which would provide the basic material for polyclonal antibody preparation, interactive factors screening and gene function research. According to the tissue-specific expression pattern analysis by q RT-PCR method, the AsMYC2 gene in A. sinensis tissues is mainly expressed in roots and stems, the main agarwood formation parts, and lowest expressed in leaves. These results indicate that AsMYC2 gene likely play some roles in agarwood formation in A. sinensis.

[Construction and expression of prokaryotic vector of AsJAZ1 gene from Aquilaria sinensis].

The full-length coding sequence (cds) of jasmonate-zim-domain protein (AsJAZ1) gene was cloned from Aquilaria sinensis, the prokaryotic vector was constructed and the recombinant proteins expression was induced to provide the basic material for interactive proteins screen and gene function research. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length cds of AsJAZ1 gene was amplified using RT-PCR method and subcloned into pET-28a vector. The recombinant plasmid identified by restriction enzyme digestion and nucleotide sequencing was transformed into E. coli BL21(DE3). Inducing with 0.5 mmol•L⁻¹ IPTG at 37 ℃ for 4 hours, a fusion protein about 39 kDa was maximumly obtained. AsJAZ1 fusion protein had been expressed successfully mainly in the form of inclusion bodies and only a very small amount was secreted into the cytoplasm in the supernatant.

[Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis].

The study aimed to clone the open reading frame of cinnamate 4-hydroxylase (C4H) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. One unique sequence containing C4H domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of C4H was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. C4H expression profiles in responds to MeJA (methyl jasmonate) application were analyzed by real-time PCR. The length of C4H open reading frame (ORF) was 1 515 bp, encoding 514 amino acids. The GenBank accession number is KF134783. Inducible-experiments showed that the genes were induced by mechanical wound as well as MeJA induction, and reached the highest expression level at 8 h and 20 h, respectively. The full-length cDNA of C4H and its expression patterns will provide a foundation for further research on its function in the molecular mechanisms of aromatic compounds and flavonoids biosynthesis.

Jasmonic acid is a crucial signal transducer in heat shock induced sesquiterpene formation in Aquilaria sinensis.

Agarwood, a highly valuable resinous and fragrant heartwood of Aquilaria plants, is widely used in traditional medicines, incense and perfume. Only when Aquilaria trees are wounded by external stimuli do they form agarwood sesquiterpene defensive compounds. Therefore, understanding the signaling pathway of wound-induced agarwood formation is important. Jasmonic acid (JA) is a well-characterized molecule that mediates a plant's defense response and secondary metabolism. However, little is known about the function of endogenous JA in agarwood sesquiterpene biosynthesis. Here, we report that heat shock can up-regulate the expression of genes in JA signaling pathway, induce JA production and the accumulation of agarwood sesquiterpene in A. sinensis cell suspension cultures. A specific inhibitor of JA, nordihydroguaiaretic acid (NDGA), could block the JA signaling pathway and reduce the accumulation of sesquiterpene compounds. Additionally, compared to SA and H2O2, exogenously supplied methyl jasmonate has the strongest stimulation effect on the production of sesquiterpene compounds. These results clearly demonstrate the central induction role of JA in heat-shock-induced sesquiterpene production in A. sinensis.