苦荞类黄酮糖基转移酶FtUFGT1的重组表达与酶学性质分析

Recombinant Expression and Catalytic Properties of Tartary Buckwheat Flavonoid Glycosyltransferase FtUFGT1

黄酮类化合物是苦荞重要的功能性成分,其糖基化修饰可改变生物体内黄酮类化合物的稳定性、可溶性及生物活性。该研究基于苦荞转录组数据并以苦荞叶片中提取的总RNA为材料,利用RT-PCR克隆了苦荞类黄酮糖基转移酶(UDP-glycose:flavonoidglycosyltransferase,UFGT)基因FtUFGT1,采用无缝克隆方式构建其重组表达载体并转化大肠杆菌Rosetta(DE3)感受态,采用GST-resin纯化重组表达的蛋白,采用高效液相色谱(HPLC)技术检测分析纯化后FtUFGT1的酶学性质。结果表明:(1)成功克隆的FtUFGT1编码区为1413 bp,其编码470个氨基酸,并成功构建了FtUFGT1的重组表达载体pGEX-6p-1-FtUFGT1。(2)经转化苦荞FtUFGT1基因在大肠杆菌Rosetta(DE3)中得到可溶性的表达,并通过GST亲和层析纯化得到高纯度的苦荞FtUFGT1蛋白。(3)HPLC分析显示,以槲皮素为底物,苦荞FtUFGT1可催化异槲皮素的合成,比活力为9.174 U/mg;重组FtUFGT1的最适温度为30℃,最适pH为7.0,5%(V/V)的甲醇和0.5%(V/V)的Triton X-100可以显著抑制其活性。研究结果为深入揭示FtUFGT1的生物学功能及体外催化黄酮类衍生物的合成奠定了基础。

Flavonoids are important functional components of tartary buckwheat,and its glycosylation modification can change the stability,solubility and biological activity of flavonoids in organisms.Based on the analysis of transcriptome data of tartary buckwheat,we cloned the tartary buckwheat flavonoid glycosyltransferase(UDP-glycose:flavonoid glycosyltransferase,UFGT)gene FtUFGT1 with RT-PCR using the total RNA extracted from tartary buckwheat leaves.The recombinant expression vector was constructed by seamless cloning method and transformed into E.coli Rosetta(DE3)competent cell.The recombinant expressed protein was purified by GST-resin,and the catalytic properties of purified FtUFGT1 were analyzed by high performance liquid chromatography(HPLC).The results showed that:(1)the cloned FtUFGT1 coding region was 1413 bp,which encodes 470 amino acids,and the recombinant expression vector pGEX-6P-1-FtUFGT1 was successfully constructed.(2)The tartary buckwheat FtUFGT1 gene expressed soluble protein in E.coli Rosetta(DE3),and high-purity FtUFGT1 was purified by GST affinity chromatography.(3)HPLC analysis showed that tartary buckwheat FtUFGT1 could catalyze the synthesis of isoquercetin with quercetin as the substrate,with a specific activity of 9.174 U/mg;the optimum temperature of recombinant FtUFGT1 is 30℃,and the optimum pH is 7.0.In addition,5%(V/V)methanol and 0.5%(V/V)Triton X-100 could significantly inhibit its activity.These results laid the foundation for further revealing the biological function of FtUFGT1 and catalyzing the synthesis of flavonoid derivatives in vitro.