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.