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通过cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)从益母草(Leonurus heterophyllus Sweet)叶片中克隆获得了一个编码糖基转移酶的基因(LhsUGT)。该基因cDNA全长为1562 bp,开放阅读框(open reading frame,ORF)为1368 bp,编码455个氨基酸残基,其分子量和等电点分别为50.47 k D和5.52。生物信息学分析结果显示:LhsUGT编码的酶蛋白含有3种二级结构,其中α螺旋占43.1%,β折叠片占17.5%,无规卷曲占39.4%,其C端还发现了一段高度保守的PSPG结构域,说明LhsUGT编码的酶蛋白属于植物糖基转移酶超家族;对LhsUGT的氨基酸序列进行BLAST同源比对,发现益母草与其它植物的糖基转移酶序列相似性为26.4%~68.0%;系统进化树分析表明,LhsUGT蛋白属于拟南芥糖基转移酶超家族的L组,故推测它可能具有催化简单酚类发生糖基化的功能;SDS-PAGE电泳显示,原核表达系统成功表达出分子量约为69 k D的LhsUGT重组蛋白,其N端含有一段17.9 k D的His标签,且在IPTG诱导5 h后达到最高表达量。本研究结果为进一步开展体外酶促反应、阐明益母草糖基转移酶的生物学功能奠定了基础。
A gene encoding glycosyltransferase (LhsUGT) was cloned from leaves of Leonurus heterophyllus Sweet by rapid amplification of cDNA ends (RACE). The full-length cDNA was 1562 bp in length and 1368 bp in open reading frame (ORF) encoding 455 amino acid residues with a molecular weight and isoelectric point of 50.47 kD and 5.52, respectively. The results of bioinformatics analysis showed that LhsUGT encoded three kinds of secondary structures, of which α-helix accounted for 43.1%, β-sheet 17.5% and random coil 39.4%. A highly conserved PSPG domain, indicating that the LhsUGT encoded enzyme belongs to the plant glycosyltransferase superfamily; BLAST homology alignment of the amino acid sequence of LhsUGT revealed that the similarity of glycosyltransferase sequences between the motherwort and other plants was 26.4% -68.0% Phylogenetic analysis showed that LhsUGT belongs to group L of the Arabidopsis glycosyltransferase superfamily, suggesting that it may catalyze the glycosylation of simple phenols. SDS-PAGE electrophoresis showed that the prokaryotic expression system was successfully expressed The LhsUGT recombinant protein with a molecular weight of about 69 kD contained a His-tag of 17.9 kD at the N-terminus and reached the highest level after IPTG induction for 5 h. The results of this study laid the foundation for the further development of the enzymatic reaction in vitro and elucidation of the biological function of the motherwort glycosyltransferase.