金银花糖基转移酶基因的原核表达及其编码蛋白纯化  

Prokaryotic expressions and encode protein purification of glycosyltransferase in Lonicerae Japonicae Flos

作　　者：蔡芷辰[1] 赵君谊 陈海杰 刘训红[1] 王进[1] CAI Zhichen;ZHAO Junyi;CHEN Haijie;LIU Xunhong;WANG Jin(Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学,江苏南京210023

出　　处：《中草药》2025年第4期1338-1345,共8页Chinese Traditional and Herbal Drugs

基　　金：江苏省基础研究计划自然科学基金资助项目(BK20220479);江苏省普通高校自然科学研究项目(22KJB360007);国家中医药管理局高水平中医药重点学科建设项目(国中医药人教函[2023]85号)。

摘　　要：目的以金银花Lonicerae Japonicae Flos中糖基转移酶为研究对象,进行糖基转移酶LjUGT73C1基因的生物信息学分析、基因合成与亚克隆、表达分析和纯化。方法利用前期盐胁迫金银花组学研究构建的金银花转录组数据库注释信息,筛选得到金银花中关键糖基转移酶LjUGT73C1,进行基因合成和亚克隆;利用基因重组技术构建了原核表达载体Pet-30a-LjUGT73C1,遗传转化大肠杆菌BL21(DE3)感受态;应用SDS-PAGE凝胶电泳和Western blotting检测蛋白的表达量、检测不同诱导条件下基因的表达情况,最后经亲和色谱进行纯化。结果通过基因合成与亚克隆得到糖基转移酶LjUGT73C1的全长序列,理论编码氨基酸数目为493,等电点(PI)预测为6.27,理论相对分子质量为55530。构建了LjUGT73C1基因的原核表达载体并且在大肠杆菌中诱导表达得到重组蛋白,应用His亲和色谱柱进行蛋白纯化并经SDS-PAGE凝胶电泳检测,确定为金银花LjUGT73C1蛋白。结论首次报道了盐胁迫金银花中糖基转移酶基因LjUGT73C1,并进行表达分析和纯化,为进一步研究金银花中苯丙素的生物合成及表达调控研究奠定了基础。Objective The LjUGT73C1 gene,which was a glycosyltransferase gene from Jinyinhua(Lonicerae Japonicae Flos,LJF),was performed bioinformatics analysis,gene synthesis with subcloning,expression analysis and purification.Methods Based on the transcriptome database annotation information of LJF constructed in the previous salt stress omics study,the key glycosyltransferase LjUGT73C1 was screened,then gene synthesis and subcloning were performed.The prokaryotic expression vector Pet-30a-LjUGT73C1 was constructed by gene recombination technology,and transformed into competent Escherichia coli BL21(DE3).SDS-PAGE gel electrophoresis and Western blot were used to detect the expression of proteins,and the expression of gene under different induction conditions was detected.Finally,the protein was purified by affinity chromatography.Results The full-length sequence of glycosyltransferase LjUGT73C1 was obtained by gene synthesis and subcloning.The theoretical number of amino acids encoded was 493,the predicted isoelectric point(PI)was 6.27,and the theoretical molecular weight was 55530.The prokaryotic expression vector of gene LjUGT73C1 was constructed and the recombinant protein was induced and expressed in E.coli.The protein was purified by His affinity chromatography and identified as LjUGT73C1 by SDS-PAGE.Conclusion The LjUGT73C1 glucosyltransferase gene in LJF under salt stress was first reported,and its expression analysis and purification were carried out.This study laid a foundation for further study on the biosynthesis and expression regulation of phenylpropanoids in LJF.

关 键 词：金银花 LjUGT73C1基因 生物信息分析 原核表达 蛋白纯化 

分 类 号：R286.12[医药卫生—中药学]