苦荞糖基转移酶基因的克隆及活性鉴定

Molecular Cloning and Expression of Glycosyltransferases Gene from Fagopyrum tataricum

糖基化修饰在调控各种小分子的溶解度、稳定性及生物活性中具有重要的作用。该研究基于苦荞转录组数据,克隆获得2条糖基转移酶基因(FtUFGT4和FtUFGT5),并对其在大肠杆菌中的表达产物进行酶催活性鉴定。结果表明:(1)获得的苦荞糖基转移酶基因cDNA分别为1 434和1 470bp,其编码蛋白同属于拟南芥糖基转移酶E类群,可能参与黄酮类化合物的糖基化。(2)多重序列比对表明,FtUFGT4和FtUFGT5蛋白C端都具有PSPG框,其催化活性位点分别是H17和H16;FtUFGT4和FtUFGT5都是典型的植物糖基转移酶GT-B结构,二者的蛋白模型能与矢车菊素和UDP进行分子对接。(3)FtUFGT4和FtUFGT5在大肠杆菌中获得了可溶性表达,薄层层析实验表明二者均具有催化矢车菊素糖基化为矢车菊素-3-O-葡萄糖苷的活性。

Glycosylation modification plays an important role in the regulation of solubility,stability and biological activity of various small molecules.Based on transcriptome data,we cloned two glycosyltransferase genes from tartary buckwheat and expressed them in E.coli.The result showed that：（1）the cDNA sequences of FtUFGT4 and FtUFGT5were 1 434 bp and 1 470 bp in length,respectively.Both of their coding proteins were classifieds E group of AtUFGTs from Arabidopsis thaliana,which may be involved in flavonoid glycosylation.（2）Multiple sequence alignment indicated that there was a PSPG Box at their C-terminal,and the catalytic activity site was H16 and H17,respectively.Meanwhile,both of them had a typical GT-B structures in plant glycosyltransferase.Moreover,the molecular docking results exhibited that FtUFGT4 and FtUFGT5could dock with cyanidin and UDP.（3）FtUFGT4and FtUFGT5 were ectopic expressed in the E.coli solubly.The thin layer chromatography（TLC）proved that they showed the cyanidin 3-O-glucoside activity.