Full-length cDNA cloning of AsJAZ1 gene and damage-induced expression analysis in Aquilaria sinensis callus / 中草药

Objective: To clone the full-length cDNA of jasmonate-zim-domain protein (JAZ) gene in Aquilaria sinensis to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of JAZ in A. sinensis was cloned through rapid amplification of cDNA ends (RACE) technique and reverse transcription PCR (qRT-PCR) method. The bioinformatics of the JAZ gene was analyzed as well. The expression of this gene was detected by qRT-PCR method with MeJA and mechanical wounding treatment in A. sinensis callus. Results: The full-length cDNA (1 507 bp) of JAZ gene was named AsJAZ1; GenBank registration number was KP677281. AsJAZ1 was obtained with an open reading frame (ORF) of 990 bp and encoding 330 amino acids. The relative molecular mass of AsJAZ1 calculated was 34 280, and the isoelecric point was 6.89. Real time PCR results indicated that both MeJA treatment and mechanical wounding could stimulate the increase of mRNA expression of AsJAZ1; There was a sharp rise at 0.5 h with about 27 times higher than the control (without MeJA treatment) with MeJA treatment, then dropped significantly. In mechanical wounding treatment, the highest peak presented in 2 h about 17 times compared to the control, then dropped significantly too. The expression of AsJAZ1 gene returned to be normal in 24 h. Conclusion: We have obtained the full-length cDNA sequence of AsJAZ1 gene firstly, which was extremely sensitive to wounding and responded to the early damage.

Construction and expression of prokaryotic vector of AsJAZ1 gene from Aquilaria sinensis / 中国中药杂志

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.

Prokaryotic expression and purification of Aquilaria sinensis (Lour.) Gilg AsMYC2 protein / 药学学报

The MYC2 transcription factor is a member of the important plant bHLH 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 qRT-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.

Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis / 中国中药杂志

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.

Full-length cDNA cloning of AsNINJA1 gene in Aquilaria sinensis and MeJA-induced expression in callus / 中草药

Objective: To clone the full-length cDNA of interacting protein of JAZ (NINJA) gene in Aquilaria sinensis, and to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of NINJA in A. sinensis was cloned through RACE technique and RT-PCR method. The bioinformatics of cloing NINJA gene was analyzed as well. The expression mode of this gene was with MeJA treatment in A. sinensis callus detected by qRT-PCR method. Results: The full-length cDNA (1 982 bp) of NINJA gene in A. sinensis, named as AsNINJA1 was obtained with an open reading frame of 1 221 bp and encoding 406 amino acids. The relative molecular mass of AsNINJA1 protein calculated was 43 697, and the isoelectric point was 6.02. The qRT-PCR results indicated that MeJA treatment could stimulate the increase of mRNA expression of AsNINJA1; There was a sharp rise at 4 h with nearly 100 times higher than the control (without MeJA treatment), then dropped significantly. Conclusion: The full-length cDNA sequence of AsNINJA1 gene is obtained; AsNINJA1 is extremely sensitive to MeJA treatment, and responded to the early damage.