山葡萄类黄酮-3-O-葡萄糖基转移酶基因的表达分析  

Analysis of Expression of Flavonoid-3-O-Glucosyltransferase Gene in Vitis amurensis

作　　者：陈蒙 袁赫海 杨铭慧 张宇[1] 刘海峰[1] CHEN Meng;YUAN Hehai;YANG Minghui;ZHANG Yu;LIU Haifeng(Agricultural College of Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学农学院,吉林延吉133002

出　　处：《河南农业科学》2018年第11期95-98,共4页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(31260067);吉林省教育厅"十三五"科学技术研究项目(吉教科合字[2016]第255号);延边大学大学生创新创业训练资助项目

摘　　要：为了探究VAm3GT蛋白表达和在山葡萄体内酶学特征及生物学功能,进一步证实克隆所获得目的基因的准确性。利用实时定量PCR法对山葡萄果皮8个不同转色时期类黄酮-3-O-葡萄糖基转移酶基因(VAm3GT)的表达量进行了分析,并通过双酶切、连接转化等方法将VAm3GT基因克隆到原核表达载体pET28a上,构建原核表达重组质粒pET28a-VAm3GT。重组质粒转化至E.coli BL21后经不同浓度的IPTG诱导,通过SDS-PAGE分析表明,在适宜的IPTG诱导浓度下,VAm3GT基因在大肠杆菌BL21中得到高效表达,表达的融合蛋白分子质量约为50.19 ku,成功构建其原核表达载体并使其在大肠杆菌中得到高效表达。In order to explore the expression of VAm3GT protein and its enzymatic and biological functions in Vitis amurensis,to confirm the accuracy of the target gene obtained by cloning.The quantitative expression of flavonoid-3-O-glucosaminyltransferase(VAm 3 GT)genes in 8 different coloration stages of Vitis amurensis were analyzed by real-time quantitative PCR,and the flavonoids of Vitis amurensis were obtained by double enzyme digestion and ligation and transformation.The flavonoid-3-O-glucosyltransferase gene was cloned into the prokaryotic expression vector pET28a to construct a prokaryotic expression recombinant plasmid pET28a-VAm 3 GT.The recombinant plasmid was transformed into E.coli BL21 and induced with different concentrations of IPTG.SDS-PAGE analysis showed that the VAm 3 GT gene was highly expressed in E.coli BL21 at the appropriate concentration induced by IPTG,and the expressed fusion protein had a molecular weight of about 50.19 ku.Successfully constructed its prokaryotic expression vector and made it highly expressed in E.coli.

关 键 词：山葡萄 山葡萄类黄酮-3-O-葡萄糖基转移酶基因 实时荧光定量 原核表达 

分 类 号：S663.1[农业科学—果树学]