摘要
本文对白及中催化天麻素合成的糖基转移酶进行了功能表征,利用生物信息学方法分析其氨基酸序列、二级结构、理化性质等,RT-qPCR分析基因的组织特异性表达特征。基于白及和拟南芥中UGT蛋白序列,使用MEGA软件构建UGT蛋白的系统发育树,构建pET-28a-BsUGT28原核表达载体,将其导入BL21(DE3)感受态细胞中进行异源表达,通过纯化蛋白及体外酶活以表征其功能。进化树结果显示,BsUGT28蛋白与红景天RsUGT73B6蛋白亲缘关系较近,同源性最高并在同一分支。它的开放阅读框长1419 bp,编码蛋白含472个氨基酸,分子质量为52.57 ku,理论等电点为5.48。RT-qPCR结果显示,BsUGT28在根中高表达。成功构建了BsUGT28基因的原核表达载体,并在250 mmol/L咪唑洗脱液中得到可溶性蛋白,且其大小与预测值一致。体外酶活检测发现BsUGT28能够催化对羟基苯甲醇生成天麻素。
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 1419 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.
作者
马迪娜
刘启泽
冯垒
向贵生
张广辉
MA Dina;LIU Qize;FENG Lei;XIANG Guisheng;ZHANG Guanghui(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;National-Local Joint Engineering Research Center on Germplasm Innovation&Utilization of Chinese Medicinal Materials in Southwestern China,Yunnan Agricultural University,Kunming 650201,China;The Key Laboratory of Medicinal Plant Biology of Yunnan Province,Yunnan Agricultural University,Kunming 650201,China;Yunnan Characteristic Plant Extraction Laboratory,Kunming 650106,China)
出处
《西北农业学报》
北大核心
2025年第10期1867-1875,共9页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家重点研发课题(2022YFD1601810,2023TMXJZJ01)。
关键词
白及
糖基转移酶
基因克隆
原核表达
体外酶活
Bletilla striata
Glycosyltransferase
Gene cloning
Prokaryotic expression
In vitro en-zyme activity
