金荞麦黄酮醇合酶基因的克隆及在大肠杆菌中的表达

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1008 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.

Cloning of flavonol synthase gene from Fagopyum dibotrys and its expression in Escherichia coli

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.