鱼腥草糖基转移酶基因UGT75C1的克隆及原核表达  

Cloning and Prokaryotic Expression of Anthocyanidin 3-O-glucoside 5-O-glucosyltransferase Gene in Houttuynia cordata

作　　者：黎晓英[1] 伍贤进[1] 姚元枝[1] 付明[1] 宁鹏飞[1] 李昭君[1] 魏麟[1] 

机构地区：[1]民族药用植物资源研究与利用湖南省重点实验室,湘西药用植物与民族植物学湖南省高校重点实验室,怀化学院生物与食品工程学院,湖南怀化418008

出　　处：《园艺学报》2015年第11期2299-2305,共7页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(30870230);湖南省科技计划重点项目(2013FJ6090;2014FJ4207);湖南省教育厅创新平台开放基金项目(15K100);湖南省生物类专业大学生创新训练中心资助项目

摘　　要：根据已经获得的鱼腥草UGT75C1转录本序列设计1对引物,采用RT-PCR方法获得UGT75C1基因cDNA序列,并对UGT75C1蛋白进行理化性质分析,并预测该蛋白功能;利用实时荧光定量PCR方法检测了UGT75C1基因在鱼腥草的地下茎、地上茎、叶、花中的表达情况,将克隆得到的UGT75C1基因完整开放阅读框连接到原核表达载体pGEX4T-1上,转化大肠杆菌E.coli BL21(DE3),通过IPTG诱导表达,SDS-PAGE检测表达产物。克隆获得的UGT75C1基因长为1 787 bp,开放阅读框1 461 bp,编码486个氨基酸。生物信息学预测UGT75C1蛋白含跨膜区,不含信号肽,具有糖基转移酶的PSPG motif。UGT75C1在鱼腥草的叶片中表达丰度最高,其他器官中表达量相对较低,花中表达量最低;该基因原核表达产物与预期大小一致,显示原核表达成功,为下一步研究其功能奠定了基础。To clone the anthocyanidin 3-O-glucoside 5-O-glucosyltransferase（UGT75C1） gene from Houttuynia cordata and analyse the prokaryotic expression.The cloning primers were designed based on the transcriptome dataset of H.cordata,one unique sequence encoding UGT75C1 was discovered.The sequence of UGT75C1 was cloned from H.cordata by RT-PCR.The physical and chemical properties,secondary structure and three-dimensional structure of the UGT75C1 protein were forecasted and analyzed,and its structure and function were predicted.And the different expression levels of UGT75C1 gene in rhizome,stems,leaves,and flowers of Houttuynia cordata were analyzed by fluorescent quantitative PCR.And then the cloned opening reading frame of UGT75C1 gene was inserted into vector pGEX4T-l.The recombinant plasmid pGEX4T- UGT75C1 was expressed in a prokaryotic expression system after they were transformed into E.coli BL21（DE3）.The fusion proteins were analyzed by SDS-PAGE.The cDNA contains a 1 461 bp open reading frame and encodes a predicted protein of 486 amino acids.Transmembrane regions and no signal peptide were presented in UGT75C1.The PSPG motif domain of glycosyltransferases was presented in UGT75C1.Relative real-time PCR analysis indicated that UGT75C1 showed the highest transcript abundance in the leaves,moderate level in the stems and rhizomes,and the lowest level in the flowers.The UGT75C1 gene was expressed in a prokaryotic expression system.This study cloned the UGT75C1 gene from H.cordata for the first time.It will provide a foundation for studying the function of the UGT75C1,and it provide a scientific basis for functional genomics research of H.cordata and mechanism research of flavonoids biosynthesis.

关 键 词：鱼腥草 UGT75C1 克隆 原核表达 

分 类 号：S567.2[农业科学—中草药栽培]