目的 克隆千金藤中SjOMT31基因，并对其进行生物信息学分析及体外酶活表征来探讨其功能。方法 通过PCR扩增获得SjOMT31的全长序列，并利用生物信息学分析蛋白结构。构建PET-30a-SjOMT31原核表达载体，并将其转入BL21（DE3）大肠杆菌中进行诱导表达。蛋白纯化后进行体外酶活反应，对其功能进行表征。结果 从千金藤中扩增得到SjOMT31全长序列，生信分析发现SjOMT31为稳定亲水性蛋白，无跨膜结构域和信号肽，属于膜外蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶（sodium dodecyl sulfate - polyacrylamide gel electrophoresis，SDS-PAGE）结果分析显示，利用原核表达系统成功诱导获得可溶性SjOMT31蛋白，体外酶促反应检测结果表明，SjOMT31能够催化(S)-去甲乌药碱C6、(S)-乌药碱C7和(S)-金黄紫堇碱及四氢非洲防己碱C2位羟基甲基化。结论 成功克隆SjOMT31基因，属于膜外蛋白；同时在大肠杆菌中实现了SjOMT31的异源表达，体外酶功能表征发现SjOMT31能催化苄基异喹啉类生物碱C6、C7和原小檗碱C2位羟基发生甲基化，为其在生物碱代谢中的作用提供了重要依据。

Objective This study aims to verify the function of the SjOMT31 gene from Qianjinteng (Stephania japonica) through cloning, bioinformatics analysis and in vitro enzymatic activity assays. Methods The full-length sequence of SjOMT31 was amplified by PCR, and its protein structure was analyzed using bioinformatics tools. A prokaryotic expression vector, pET-30a-SjOMT31, was constructed and inserted into Escherichia coli BL21 cells to induce the expression of its protein. The purified protein was subjected to in vitro enzymatic activity assays to characterize its function. Results The full-length sequence of SjOMT31 was successfully cloned from S. japonica. Bioinformatics analysis revealed that SjOMT31 is a stable hydrophilic protein without transmembrane domains or signal peptides, indicating its classification as an extracellular membrane protein. SDS-PAGE results demonstrated that the soluble SjOMT31 protein was successfully induced using a prokaryotic expression system. In vitro enzymatic assays showed that SjOMT31 catalyzes the methylation of hydroxyl groups at the C6 position of (S)-norcoclaurine, the C7 position of (S)-coclaurine, and the C2 position of (S)-scoulerine and (S)-tetrahydrocolumbamine. Conclusion The SjOMT31 gene was successfully cloned and identified as an extracellular membrane protein. Heterologous expression of SjOMT31 in E. coli was achieved, and the purified protein was characterized for its in vitro enzymatic assays. SjOMT31 catalyzes the methylation of hydroxyl groups at the C6 and C7 positions of benzylisoquinoline alkaloids and the C2 position of protoberberine alkaloids. These findings provide critical insights into the role of SjOMT31 in alkaloid metabolism.

R286.12

国家自然科学基金项目（82504957）；四川省博士后创新人才支持项目（BX202206）；四川省自然科学基金青年项目（2025ZNSFSC1807）