三七中黄酮糖基转移酶的筛选与鉴定

Screening and identification of flavonoid glycosyltransferases from Notoginseng Radix et Rhizoma

目的:将前期克隆得到21条三七来源的糖基转移酶基因,构建到pCold表达载体,以实现重组蛋白的可溶性表达,并筛选、鉴定其在体外对黄酮类化合物的催化功能。方法:对21条糖基转移酶基因进行系统进化树分析;利用同源重组的方法将其克隆到pCold表达载体上,并在大肠杆菌中表达重组蛋白。低温诱导重组蛋白表达,以黄酮类化合物为糖基受体底物,UDP-葡萄糖为糖基供体,采用高效液相色谱法(HPLC)检测酶促反应产物。结果:分别以山柰酚和芹菜素为底物,重组蛋白PnUGT82参与反应后,可检测到山柰酚7-O葡萄糖苷和芹菜素7-O葡萄糖苷,而阴性对照组未检测到对应的糖苷产物。结论:筛选得到1条三七来源的糖基转移酶PnUGT82,能够催化黄酮类化合物,在体外将UDP-葡萄糖上的葡萄糖基团转移到山柰酚或芹菜素7位羟基,形成相应的黄酮7-O糖苷化产物。

Objective: Twenty-one glycosyltransferase genes, cloned from Notoginseng Radix et Rhizoma in previous study, are separately constructed into pCold expression vector to realize the soluble expression of recombinant protein, and their catalytic function towards flavonoids in vitro is screened and identified. Methods: Phylogenetic tree analysis was performed using 21 glycosyltransferase genes. These genes were separately cloned into pCold expression vector by homologous recombination, and the recombinant proteins were expressed in Escherichia coli. The recombinant proteins were induced to express at low temperature. Taken flavonoids as glycosyl receptor substrates and UDP-glucose as glycosyl donor, the enzymatic reaction products were detected by high performance liquid chromatography(HPLC). Results: Using kaempferol and apigenin as substrates respectively, kaempferol 7-O glucoside and apigenin 7-O glucoside could be detected after the recombinant protein PnUGT82 participated in the reaction;while the corresponding glycoside products were not detected in the negative control group. Conclusion: A glucosyltransferase PnUGT82 from Notoginseng Radix et Rhizoma is obtained, which can catalyze flavonoids. The glucosyl from UDP-glucose is transferred to the 7-hydroxyl of kaempferol or apigenin, to form the corresponding flavonoid 7-O glycoside products in vitro.