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

ABSTRACT

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.