目的 AsMAPK1和AsMAPK2是白木香Aquilaria sinensis中2个功能未知的丝裂原活化蛋白激酶基因，为探究其基本特性及其在伤害诱导沉香形成中潜在功能，对其进行克隆、表达分析及初步体外功能检测。方法 以课题组继代培养的白木香愈伤组织cDNA为模板，PCR扩增获得AsMAPK1和AsMAPK2的编码序列；利用相关在线工具及软件进行生物信息学分析；构建pGEX4T-1-AsMAPK1/2原核表达载体，转化大肠杆菌BL21表达并对诱导条件进行优化；构建AsMAPK1-GFP和AsMAPK2-GFP融合表达载体，转化拟南芥原生质体后利用共聚焦显微镜观察其亚细胞定位；采用体外磷酸化方法检测纯化蛋白的激酶活性。结果 成功克隆了AsMAPK1（1 188 bp）和AsMAPK2（1 128 bp）基因。生物信息学分析显示，二者编码的蛋白均含有典型的激酶结构域，属于MAPK家族成员。系统进化分析表明，AsMAPK1与拟南芥中的AtMAPK6同源蛋白亲缘关系最近，属于A组，而AsMAPK2则与B组的AtMAPK13聚为一支。SDS-PAGE检测显示，成功诱导表达了约71 000和69 000的可溶性重组蛋白。亚细胞定位结果表明，AsMAPK1-GFP和AsMAPK2-GFP都同时定位于细胞质和细胞核。进一步的激酶活性分析证实，AsMAPK1和AsMAPK2均能发生自磷酸化，并能磷酸化通用底物MBP。结论 证实AsMAPK1和AsMAPK2是同时定位于细胞质和细胞核、具有活性的蛋白激酶，通过对这2个基因的克隆、表达与分析，为深入解析其参与白木香伤害诱导形成沉香的信号传导机制奠定基础。

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.

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国家自然科学基金项目（82173925）；中国医学科学院医学与健康科技创新工程—重大协同创新项目（2021-I2M-1-032）