药材与资源金荞麦黄酮醇合酶基因的克隆及在大肠杆菌.PDFVIP

中草药 Chinese Traditional and Herbal Drugs 第 43 卷 第 14 期 2013 年 7 月 ·1974· ? 药材与资源 ? 金荞麦黄酮醇合酶基因的克隆及在大肠杆菌中的表达 蒋 洁，白悦辰，李成磊，陈 惠，吴 琦* 四川农业大学生命科学与理学院，四川 雅安 625014 摘 要：目的 克隆金荞麦黄酮醇合酶（FdFLS）基因的编码序列，并对该基因进行序列分析、原核表达及其活性研究。方 法 采用同源克隆技术获得 FdFLS 基因 cDNA 序列，并对其进行生物信息学分析；构建 FdFLS 原核表达载体 pET-30b (＋)-FdFLS，在大肠杆菌 BL21（DE3）中诱导表达，并测定目标蛋白的酶学活性。结果 FdFLS 基因开放阅读框（ORF） 全长 1 008 bp，编码 334 个氨基酸；FdFLS 基因在大肠杆菌中可表达出相对分子量约 4×104的蛋白，并具有将二氢槲皮素和 二氢山柰酚转化为槲皮素和山柰酚的催化活性。结论 首次克隆到 FdFLS 基因，并实现该基因在大肠杆菌中有活性的表达。 关键词：金荞麦；黄酮醇合酶；基因克隆；原核表达；序列分析 中图分类号：R282.12 文献标志码：A 文章编号：0253 - 2670(2013)14 - 1974 - 05 DOI: 10.7501/j.issn.0253-2670.2013.14.020 Cloning of flavonol synthase gene from Fagopyum dibotrys and its expression in Escherichia coli JIANG Jie, BAI Yue-chen, LI Cheng-lei, CHEN Hui, WU Qi College of Life Science, Sichuan Agricultural University, Ya’an 625014, China Abstract: Objective To clone the coding sequence of flavonol synthase gene from Fagopyum dibotrys (FdFLS) and to analyze its sequence, prokaryotic expression, and activity. Methods The cDNA sequence of FdFLS gene was obtained by homology cloning and analyzed by bioinformatic method; The FdFLS prokaryotic expression vector pET-30b (+)-FdFLS was established and induced to express in Escherichia coli BL21 (DE3), as well as the enzymatic activity of the target protein was measured. Results The full length of open reading frame (ORF) of FdFLS gene was 1 008 bp that encoded a protein of 334 amino acids; The recombinant FdFLS protein had a relative molecular mass of 40 000. It also has a catalytic activity, which could make dihydroquercetin and dihydrokaempferol into quercetin and kaempferol, respectively. Conclusion The FdFLS gene is successfully cloned and reported for the first time, which has an active expression in E. coli. Key words: Fagopyum dibotrys (D. Don) Hara; flavonol synthase; gene cloning; prokaryotic expression; sequence analysis 多年生草本植物金荞麦 Fagopyum dibotrys (D. Don) Hara 属双子叶蓼科（Polyg