目的 对绞股蓝Gynostemma pentaphyllum糖基转移酶基因GpUGT74A2进行克隆，并对其进行生物信息学分析及催化功能验证。方法 从绞股蓝的转录组学数据中筛选获得GpUGT74A2基因；通过在线分析网站对该序列进行生物信息学分析，运用MEGA 11软件构建系统发育树；利用大肠杆菌体外表达，高效液相色谱检验该酶对底物20(S)-原人参二醇 [20(S)-protopanaxadiol，PPD]、20(S)-原人参三醇 [20(S)-protopanaxatriol，PPT] 是否有催化活性。结果 通过PCR扩增对GpUGT74A2基因进行全长克隆（GenBank：PQ272699.1），该基因的CDS序列为1 386 bp，编码461个氨基酸。GpUGT74A2蛋白相对分子质量为52 248，理论等电点为6.49，无信号肽和跨膜区，具有端结构域PSPG（putativesecondary plantglycosyltransferase）结构域。比对已报道具有催化人参皂苷活性的氨基酸序列，显示GpUGT74A2蛋白与拟南芥中UGT76E11蛋白的同源性最高，为46.58%。色谱结果显示GpUGT74A2蛋白对PPT具有显著的催化活性，表明该基因参与绞股蓝皂苷生物合成，提出绞股蓝来源糖基转移酶有望成为稀有人参皂苷定向生物合成新的工具酶。结论 成功克隆获得GpUGT74A2基因，并进行生物信息学分析和体外催化功能验证。为进一步探究GpUGT74A2基因功能及绞股蓝来源糖基转移酶成为稀有人参皂苷定向生物合成新的工具酶提供实验依据。

Objective To clone the glycosyltransferase gene GpUGT74A2 from Gynostemma pentaphyllum and perform bioinformatics analysis to verify its catalytic function. Methods The GpUGT74A2 gene was identified from the transcriptomic data of G. pentaphyllum. Bioinformatics analysis of the sequence was performed using online analysis website, and a phylogenetic tree was constructed using MEGA 11 software. The gene was expressed in Escherichia coli, and high-performance liquid chromatography (HPLC) was used to assess the enzyme’s catalytic activity toward substrates 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT). Results Full-length cloning of GpUGT74A2 (GenBank: PQ272699.1) was achieved through PCR amplification. The coding sequence CDS (CDS) of GpUGT74A2 is 1 386 bp long, encoding 461 amino acids. The encoded protein has a relative molecular weight of 52 248 and a theoretical isoelectric point of 6.49. Structural analysis showed that the protein lacked signal peptides and transmembrane regions but contains a plant secondary product glycosyltransferase (PSPG) domain. Sequence comparison with reported amino acid sequences involved in ginsenoside catalysis revealed that GpUGT74A2 shared the highest homology (46.58%) with the UGT76E11 protein from Arabidopsis thaliana. Chromatographic analysis revealed that GpUGT74A2 exhibited significant catalytic activity toward PPT, indicating its role in the biosynthesis of G. pentaphyllum saponins. It was proposed that the glycosyltransferase derived from G. pentaphyllum was expected to become a new tool enzyme for the targeted biosynthesis of rare ginsenosides. Conclusion GpUGT74A2 has been successfully cloned, and both bioinformatics analysis and in vitro catalytic function verification have been conducted. This study provides experimental evidence for further exploration of its function and G. pentaphyllum derived glycosyltransferase to become a new tool enzyme for the directed biosynthesis of rare ginsenosides.

R286.12

国家自然科学基金资助项目(82104321); 中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”(2060302); 福建中医药大学基础类学科科研提升计划项目(XJC2023009);福建中医药大学校管重点项目(X2022003)