苦荞黄酮醇合酶FtFLS2的重组表达及多克隆抗体制备

Prokaryotic Expression of Tartary Buckwheat Flavonol Synthase FtFLS2 and Preparation of Its Polyclonal Antibody

黄酮醇合酶催化二氢黄酮醇生成黄酮醇,是黄酮类物质代谢途径中的关键酶。为深入研究苦荞黄酮醇合酶在苦荞黄酮类物质代谢中的功能,本研究采用同源克隆技术获得苦荞黄酮醇合酶基因Ft FLS2,并构建原核表达载体p ET47b-Ft FLS2;对经钴离子螯合层析柱纯化的重组蛋白进行活性分析并制备多克隆抗体。结果表明,Ft FLS2在大肠杆菌BL21 Star(DE3)中以可溶性形式高效表达,重组蛋白具有将二氢槲皮素转化为槲皮素的催化活性;Western blotting结果显示,Ft FLS2主要在苦荞未成熟种子中表达。多克隆抗体的制备为从蛋白表达水平上分析Ft FLS2与苦养黄酮类化合物累积的关系奠定了基础。

Flavonol synthase,catalysing dihydroflavonol to flavonol,play a key role in flavonoids metabolic pathway. In order to study the function of flavonol synthase in flavonoids metabolism of tartary buckwheat,Tartary buckwheat flavonol synthase gene Ft FLS2 was cloned by homology-based cloning. and a prokaryotic expression vector p ET47b-Ft FLS2 was constructed The overexpressed protein was purified by cobalt chelating chromatography and served as antigen for polyclonal antibody preparation and for enzymatic activity assay. The results showed that the recombinant Ft FLS2 protein was expressed in a soluble form in E. coli BL21 Star（ DE3） and had the catalytic activity to convert dihydroquercetin to quercetin. Western blotting results indicated that Ft FLS2 was mainly expressed in immature seeds of tartary buckwheat.These results laid the foundation for further revealing the relationships between Ft FLS2 expression level and accumulation of flavonoids in Tartary Buckwheat.