西北农业学报2025，Vol.34Issue(10)：1867-1875,9.DOI:10.7606/j.issn.1004-1389.2025.10.010
白及中天麻素合成酶基因克隆与功能表征
Cloning and Functional Characterization of Gastrodin Synthase Gene in Bletilla striata
摘要
Abstract
This study functionally characterized a glycosyltransferase(BsUGT28)involved in gastrod-in synthesis in Bletilla striata,employing bioinformatics methods to analyze its amino acid sequence,secondary structure,and physicochemical properties.The tissue-specific expression profile of the gene encoding this enzyme was examined using RT-qPCR.Based on the UGT protein sequences from Bletilla striata and Arabidopsis thaliana,a phylogenetic tree of the UGT family was constructed using MEGA software.The prokaryotic expression vector pET-28a-BsUGT28 was constructed and in-troduced into BL21(DE3)competent cells for heterologous expression.The purified protein was ob-tained for functional characterization through in vitro enzymatic assays.The phylogenetic tree indica-ted a high similarity between the BsUGT28 protein and the Rhodiola rosea RsUGT73B6 protein,as they exhibited the highest homology and clustering on the same branch.The open reading frame of BsUGT28 is 1 419 bp long,encoding a protein composed of 472 amino acids,with a molecular mass of 52.57 ku and a theoretical isoelectric point of 5.48.RT-qPCR results indicated that BsUGT28 is high-ly expressed in the roots.The prokaryotic expression plasmid of the BsUGT28 gene was successfully constructed,and a soluble protein of the expected size was obtained in 250 mmol/L imidazole elution buffer,with consistent size as predicted.In vitro enzyme activity assays revealed that BsUGT28 could convert p-hydroxybenzyl alcohol to gastrodin.关键词
白及/糖基转移酶/基因克隆/原核表达/体外酶活Key words
Bletilla striata/Glycosyltransferase/Gene cloning/Prokaryotic expression/In vitro en-zyme activity引用本文复制引用
马迪娜,刘启泽,冯垒,向贵生,张广辉..白及中天麻素合成酶基因克隆与功能表征[J].西北农业学报,2025,34(10):1867-1875,9.基金项目
国家重点研发课题(2022YFD1601810,2023TMXJZJ01). National Key R&D Plan(No.2022YFD1601810,No.2023TMXJZJ01). （2022YFD1601810,2023TMXJZJ01）
