摘要
目的 克隆知母糖基转移酶基因UGT708Z1,对其进行生物信息学、原核表达分析以及功能鉴定。方法 基于转录组数据,从知母中挖掘并筛选到一条候选糖基转移酶基因UGT708Z1。根据其全长开放阅读框设计带有同源臂的特异性引物,采用PCR进行基因克隆;通过同源重组技术构建pET-32a(+)-UGT708Z1重组质粒,并利用原核表达和纯化蛋白技术获得可溶性目的蛋白。最后,采用体外酶促反应对UGT708Z1进行功能鉴定。结果 序列分析表明UGT708Z1的开放阅读框为1377 bp,编码458个氨基酸。原核表达结果显示,UGT708Z1成功表达出可溶性目的蛋白,纯化后重组蛋白大小为70.86 kDa。体外酶促反应结果表明,UGT708Z1具有类黄酮7-OH糖基化活性,能够催化淫羊藿素生成淫羊藿次苷Ⅰ。此外,UGT708Z1还具有催化槲皮素和芹菜素的7-O-糖基化活性的功能。结论 本研究从知母中成功克隆并鉴定出一个黄酮醇糖基转移酶UGT708Z1,为后续深入解析黄酮醇苷的生物合成奠定基础。
Objective To clone the glycosyltransferase gene UGT708Z1 in Anemarrhena asphodeloides and perform its bioinformatics analysis,prokaryotic expression analysis and functional characterization.Methods A candidate glycosyltransferase gene UGT708Z1 was mined and screened out from Anemarrhena asphodeloides based on the transcriptome data.According to its full-length open reading frame,the specific primers with homologous arms were designed.Subsequently,the UGT708Z1 gene was cloned by PCR.The prokaryotic expression recombinant vector pET-32a(+)-UGT708Z1 was constructed through homologous recombination technology,and the soluble target protein was obtained by prokaryotic expression and purified protein technology.Finally,the function of UGT708Z1 was identified through enzymatic reaction in vitro.Results Sequence analysis showed that the open reading frame of UGT708Z1 was 1377 bp,encoding 458 amino acids.The result of prokaryotic expression showed that UGT708Z1 successfully expressed the soluble target protein,and the purified recombinant protein was 70.86 kDa.The results of enzymatic reaction in vitro showed that UGT708Z1 had flavonoid 7-OH glycosylation activity and could catalyze icaritin to produce icariside I.In addition,UGT708Z1 also possessed the activities of catalyzing the 7-O-glycosylation of quercetin and apigenin.Conclusion In this study,a flavonol glycosyltransferase UGT708Z1 was successfully cloned and identified from Anemarrhena asphodeloides,which would lay a foundation for further analysis of flavonol glycosides biosynthesis.
作者
张倩
纪中菊
李志新
董子舒
刘红宁
王晓云
黄佳
ZHANG Qian;JI Zhongju;LI Zhixin;DONG Zishu;LIU Hongning;WANG Xiaoyun;HUANG Jia(Institute for Advanced Study,Research Center of Chinese Medicine Resource and National Medicine,Jiangxi University of Chinese Medicine,Nanchang 330004,China;International Institute for Translational Chinese Medicine,School of Pharmaceutical Sciences,Guangzhou University of Traditional Chinese Medicine,Guangzhou 510000,China)
出处
《世界科学技术-中医药现代化》
北大核心
2025年第10期2800-2809,共10页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
江西省科学技术厅青年基金项目(20242BAB20452):中药活性成分知母皂苷BII生物合成的糖基化分子机制研究
负责人:黄佳。
关键词
知母
糖基转移酶
基因克隆
原核表达
功能表征
Anemarrhena asphodeloides
Glycosyltransferase
Gene cloning
Prokaryotic expression
Functional characterization
