Gene Cloning and Prokaryotic Expression of Glycosyltransferase from Leonurus heterophyllus Sweet(英文稿)
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摘要: 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1562bp,开放阅读框(openreading frame,ORF)为1368bp,编码455个氨基酸残基,其分子量和等电点分别为50.47kD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69kD的LhsUGT重组蛋白,其N端含有一段17.9kD的His标签,且在IPTG诱导5h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。Abstract: A glycosyltransferase gene (LhsUGT) was cloned from Leonurus heterophyllus Sweet using rapid amplification of cDNA ends (RACE). The full length cDNA of LhsUGT was 1562 bp, with a 1368 bp open reading frame (ORF) encoding a 455 amino-acid protein (LhsUGT) and a molecular weight of 50.47 kD and isoelectric point of 5.52. By bioinformatics analysis, three secondary structures were discovered in LhsUGT, including 43.1% helix, 17.5% β-sheet and 39.4% random coil structural elements. There was a conserved PSPG domain at the C-terminal of LhsUGT, demonstrating that the enzyme encoded by LhsUGT belonged to the glucosyltransferase super family. Homology analysis by BLAST showed that the sequence similarities of glycosyltransferase between L. heterophyllus and other plants were 26.4% - 68.0%. Phylogenetic tree analysis indicated that LhsUGT might glycosylate simple phenolic compounds as it was found to be clustered with group L in Arabidopsis thaliana. SDS-PAGE analysis showed recombinant LhsUGT with approximate molecular weight of 69 kD was successfully expressed by a prokaryotic expression system, which contained His tags of 17.9 kD at the N-terminal and reached maximum expression level until 5 h after induction by IPTG (Isopropyl β-D-1-thiogalactopyranoside). The results in the study will lay the foundation for future studies on the biological functions of LhsUGT by enzymatic reactions in vitro.
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