摘要
【目的】克隆苦荞FtFLS4基因,深入研究其酶学活性。【方法】根据苦荞转录组数据克隆FtFLS4基因,对其进行生物信息学分析;构建原核表达载体pET-30b(+)-FtFLS4并在大肠杆菌BL21(DE3)中诱导表达,对表达产物进行活性鉴定。【结果】FtFLS4基因的cDNA为1 026 bp,编码341个氨基酸,编码蛋白具有属于α-ODD家族中黄酮醇合酶的结构特征,且其三维模型能与3种二氢黄酮醇底物进行分子对接;FtFLS4在大肠杆菌BL21(DE3)中实现可溶性表达,酶学分析表明该重组蛋白具有将二氢黄酮醇催化为黄酮醇的活性。【结论】克隆得到FtFLS4基因,实现其在大肠杆菌中有活性的表达。
[Objective] The aim of the study was to clone of the FtFLS4 gene of tartary buckwheat and explore its enzymatic activity. [Method] The FtFLS4 gene was cloned from the data of tartary buckwheat transcriptional group, and analyzed by bioinformatic method. The prokaryotic expression vector pET-30b （＋）-FtFLS4 was established and induced to express in E.coli BL21（DE3）, and its activity of the expression product was identified. [Result] The eDNA sequence of FtFLS4 gene was 1026 bp in length to encode a protein with 341 amino acids, the encoded protein had the structural characteristics of flavonol synthase belonging to alpha-ODD protein family, and molecular docking showed FtFLS4 could bind with three dihydroflavonol substrates. A soluble expression of FtFLS4 could be obtained in E.coli BL21（DE3）, and the recombinant protein showed the activity of catalyzing dihydroflavonols to flavonol. [Conclusion] The FtFLS4 gene was cloned, which has an active express in E.coli.
作者
李青青
张润敏
石冠蓝
姚攀锋
王晓丽
李成磊
LI Qingqing;ZHANG Runmin;SHI Guanlan;YAO Panfeng;WANG Xiaoli;LI Chenglei(College of Life Science,SichuanAgricultural University,Ya＇an 625014,Sichuan,China)
出处
《四川农业大学学报》
CSCD
北大核心
2018年第5期611-617,664,共8页
Journal of Sichuan Agricultural University
基金
国家自然科学基金(31500289)
关键词
苦荞
黄酮醇合酶
基因克隆
原核表达
活性鉴定
tartary buckwheat
flavonol synthase
gene cloning
prokaryotic expression
activity identification
