Objective AsMAPK1 and AsMAPK2 are two mitogen-activated protein kinase (MAPK) genes with unknown functions in Aquilaria sinensis. To investigate their fundamental characteristics and potential functions in wound-induced agarwood formation, in this study, we performed cloning, expression analysis, and preliminary in vitro functional assays on the two genes. Methods Using cDNA from subcultured A. sinensis callus as the template, the coding sequences of AsMAPK1 and AsMAPK2 were obtained by PCR amplification. Bioinformatics analysis was performed using relevant online tools and softwares. The prokaryotic expression vectors pGEX4T-1-AsMAPK1/2 were constructed, transformed into E. coli BL21 for expression, and induction conditions were optimized. The AsMAPK1-GFP and AsMAPK2-GFP fusion expression vectors were constructed and transformed into Arabidopsis thaliana protoplasts to observe their subcellular localization using confocal microscopy. The kinase activity of the purified proteins was detected using an in vitro phosphorylation assay. Results The AsMAPK1 (1 188 bp) and AsMAPK2 (1 128 bp) genes were successfully cloned. Bioinformatics analysis revealed that both encoded proteins contain typical kinase domains and belong to the MAPK family. Phylogenetic analysis indicated that AsMAPK1 is most closely related to the AtMAPK6 homolog in A. thaliana and belongs to Group A, while AsMAPK2 clusters with Group B AtMAPK13. SDS-PAGE detection showed successful induction of soluble recombinant proteins of approximately 71 000 and 69 000. Subcellular localization results demonstrated that both AsMAPK1-GFP and AsMAPK2-GFP are localized in the cytoplasm and nucleus. Further kinase activity analysis confirmed that both AsMAPK1 and AsMAPK2 undergo autophosphorylation and can phosphorylate the universal substrate MBP. Conclusion This study confirms that AsMAPK1 and AsMAPK2 are active protein kinases localized in both the cytoplasm and nucleus. The cloning, expression analysis, and kinase activity detection of the two AsMAPKs provide a foundation for further elucidating their roles in the signal transduction mechanism underlying wound-induced agarwood formation in A. sinensis.

Aquilaria sinensis (Lour.) Gilg  /  mitogen-activated protein kinase (MAPK)  /  cloning  /  bioinformatic analysis  /  prokaryotic expression