苦荞黄酮醇合酶基因FtFLS4的克隆及其在大肠杆菌的表达

doi:10.16036/j.issn.1000-2650.2018.05.007

摘要/Abstract

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。

关键词: 苦荞, 黄酮醇合酶, 基因克隆, 原核表达, 活性鉴定

Abstract: 【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 cDNA 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.

Key words: tartary buckwheat, flavonol synthase, gene cloning, prokaryotic expression, activity identif-ication

中图分类号:

Q785

李青青, 张润敏, 石冠蓝, 姚攀锋, 王晓丽, 李成磊. 苦荞黄酮醇合酶基因FtFLS4的克隆及其在大肠杆菌的表达[J]. 四川农业大学学报, 2018, 36(05): 611-617.

LI Qingqing, ZHANG Runmin, SHI Guanlan, YAO Panfeng, WANG Xiaoli, LI Chenglei. Cloning of Flavonolsynthase Gene FtFLS4 from Tartary Buckwheat and Expression in E. coli[J]. Journal of Sichuan Agricultural University, 2018, 36(05): 611-617.

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