女贞叶中一条糖基转移酶的基因克隆、生物信息学分析及原核表达

Gene Cloning,Bioinformatics Analysis,and Prokaryotic Expression of A Glycosyltransferase from Ligustrum lucidum Ait.Leaves

女贞叶化学成分复杂,糖苷类物质是其发挥药理活性的关键成分,糖苷的形成离不开糖基转移酶。利用cDNA末端快速扩增技术,对女贞叶中一条糖基转移酶编码基因LlUGT3进行了克隆;根据基因和蛋白质序列进行了生物信息学分析;通过基因工程技术使LlUGT3基因在大肠杆菌体内进行了异源表达。结果表明,LlUGT3基因cDNA全长1684 bp,由85 bp 5′-UTR、159 bp 3′-UTR、1440 bp ORF组成,其中ORF编码一条含479个氨基酸残基的酶蛋白,其相对分子质量、理论等电点和亲水性平均系数分别为54.8 kDa、5.86和-0.381;在LlUGT3酶蛋白一级结构中含有一段糖基转移酶高度保守的PSPG盒,但不含信号肽且无跨膜片段,在二级结构中含有α螺旋(42.59%)、β折叠(12.73%)和无规卷曲(44.68%),在三级结构中由肽链折叠形成α/β/α类Rossmann折叠区域,并且两者之间夹着一个底物结合腔;同源建模结果显示,LlUGT3酶蛋白与模板分子PaGT3序列相似度很高,而且系统发育树分析也表明两者亲缘关系最近;分子对接分析表明,LlUGT3酶蛋白的His17-Asp116对发挥催化功能,PSPG盒主要涉及结合UDPG,而LIUGT酶与辣椒素的结合方式与PaGT3相似;通过基因工程技术,实现了LlUGT3基因在大肠杆菌体内的异源表达。该研究为进一步深入研究LlUGT3酶蛋白的结构与功能奠定了基础。

The chemical composition of Ligustrum lucidum Ait.leaves is complex,of which glycosides are the key components to exert pharmacological activities.The formation of glucoside is inseparable from glycosyltransferase.In this paper,we cloned a glycosyltransferase encoding gene LlUGT3 from Ligustrum lucidum Ait.leaves by using rapid amplification of cDNA ends.Moreover,we performed bioinformatics analysis based on gene and protein sequences.Furthermore,we performed heterologous expression of the LlUGT3 gene in Escherichia coli through genetic engineering technology.The results show that the full-length cDNA of the LlUGT3 gene is 1684 bp,consisting of 85 bp 5′-UTR,159 bp 3′-UTR,and 1440 bp ORF,in which the ORF encodes an enzyme containing 479 amino acid residues with relative molecular weight,theoretical isoelectric point,and hydrophilicity average coefficient of 54.8 kDa,5.86,and-0.381,respectively.The primary structure of the LlUGT3 enzyme protein contains a highly conserved PSPG box of glycosyltransferase,while no signal peptide and no transmembrane segment.The secondary structure containsαhelix(42.59%),βfold(12.73%),and random curl(44.68%).In the tertiary structure,two face-to-faceα/β/αRossmann folded regions are formed by folding peptide chains,with a substrate binding cavity sandwiched between them.Homologous modeling results show that the sequence of LlUGT3 enzyme protern is highly similar to that of template molecule PaGT3,and the phylogenetic tree analysis also shows that the relationship between them is the closest.Molecular docking analysis shows that the His17-Asp116 pair of LlUGT3 enzyme protein plays a catalytic function,and the PSPG box is mainly involved in binding UDPG,while LlUGT3 enzyme binds to capsaicin in a manner similar to that of PaGT3.The heterologous expression of LlUGT3 gene in E.coli is realized by genetic engineering technology.This study lays a foundation for further study on the structure and function of the LlUGT3 enzyme.