目的 揭示甜菊糖苷糖基化关键酶SrUGT76G1的构效关系。方法 采用RT-PCR法克隆各甜叶菊Stevia rebaudiana材料中的SrUGT76G1变异序列；采用原核表达系统—大肠杆菌和体外催化反应验证各变异序列功能；采用Modeller 9.17软件对SrUGT76G1各变异序列蛋白进行同源建模，利用AutoDock Vina工具进行对接分析。结果 除变异序列N02序列和N03序列外，还分别从110和B1188材料中获取了2个变异序列110-3序列和B1188序列。各变异序列蛋白体外催化反应和催化效率比较分析结果表明，N02蛋白相较于UGT76G1（N01-1，AY345974.1）活性显著下降，特别是以甜茶苷、甜菊糖苷（stevioside，ST）和莱鲍迪苷D（rebaudioside D，RD）为糖基受体底物时，B1188、N03和110-3蛋白对糖苷的糖基化功能已丧失；同源建模和分子对接分析发现由于各变异序列蛋白中氨基酸残基的改变，导致翻译形成的蛋白三维空间结构改变，导致无法完全包裹催化底物。结论 SrUGT76G1形成的活性口袋能否较好地包裹糖基受体底物是其活性强弱和有无的关键。

Objective To reveal the structure-activity relationship of SrUGT76G1, which was a key glycosyltransferase involving the glucosylation of steviol glycosides. Methods RT-PCR was used to clone the SrUGT76G1 from different Stevia rebaudiana materials. The prokaryotic expression system Escherichia coli and in vitro catalytic reaction were used to verify the function of the SrUGT76G1 variants. Modeller 9.17 software was used to model the homology of SrUGT76G1 variants, and the docking analysis was performed with AutoDock Vina tool. Results In addition to N02 and N03, two variants 110-3 and B1188 were obtained from 110 and B1188 materials respectively. Compared with UGT76G1 (N01-1, AY345974.1), the activity of N02 protein was significantly lower than that of SrUGT76G1, especially using rubusoside, stevioside (ST) and rebaudioside D (RD) as receptor substrates. And the enzyme activities of B1188, N03 and 110-3 variants were lost. Homology modeling and molecular docking analysis showed that the change of amino acid residues in each variant resulted in the change of the three-dimensional structure of the SrUGT76G1 protein, resulting in the failure to completely wrap the substrates. Conclusion Whether the active pocket formed by SrUGT76G1 can wrap the receptor substrate well is the key to its activity.

R286.12

国家自然科学基金资助项目（31671757）；西南民族大学人才类（自科）-引进人才科研启动金资助项目（16011211073）