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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。
Objective To clone the coding sequence of FdFLS gene and analyze the sequence of the FdFLS gene in E. coli. Methods The cDNA sequence of FdFLS gene was obtained by homologous cloning and bioinformatics analysis. The prokaryotic expression vector pET-30b (+) - FdFLS was constructed and expressed in E. coli BL21 (DE3). The target protein Enzymatic activity. Results The ORF of FdFLS gene was 1 008 bp in length and encoded a polypeptide of 334 amino acids. FdFLS gene expressed a protein with a molecular weight of about 4 × 104 in E. coli. The catalytic activity of 柰 phenol conversion to quercetin and kaempferol. Conclusion The FdFLS gene was cloned for the first time and the expression of this gene was confirmed in E. coli.