Prokaryotic expression of Aquilaria sinensis AsMAPK3 gene and its localization in transient expression system in onion epidermis / 中草药

ABSTRACT

Objective To construct a prokaryotic vector of AsMAPK3 gene from Aquilaria sinensis, the original plant of agarwood, and induce the recombinant proteins expression so as to study the subcellular localization of AsMAPK3. This work will prepare materials for antibody preparation and lay a foundation for screening the interaction proteins and further studying their functions. Methods Partial cDNA sequence was amplified by PCR and recombined to pET-28a vector to construct a prokaryotic expression vector pET-28a-AsMAPK3, and induced the expression of the fusion protein. The full-length cDNA of AsMAPK3 was amplified and subcloned to pAN580 vector to construct a pAN580-AsMAPK3 transient expression vector. The recombinant plasmid of pAN580-AsMAPK3 was introduced into the onion epidermis by gold particle bombardment, and GFP fluorescence was observed by luorescence microscope. Results The Escherichia coli BL21 (DE3) containing the recombinant plasmid was induced with 0.5 mmol/L isopropyl-β-D-galactoside (IPTG) at 37 ℃ for 4 h, and a fusion protein about 39 000 was obtained which was expressed in supernatant and inclusion bodies. The results of GFP fluorescence observation of transient transformed onion epidermis showed that AsMAPK3 was mainly expressed in the nucleus and plasma membrane. Conclusion The expression and purification of AsMAPK3 in vitro were successfully carried out, and the subcellular localization of AsMAPK3 gene was confirmed. This work provides a substantial foundation for follow-up function study of AsMAPK3.