女贞糖基转移酶基因(LlUGT)的克隆、生物信息学分析及原核表达

Cloning, bioinformatics analysis and prokaryotic expression of glycosyltransferase gene from Ligustrum lucidum (LlUGT)

根据前期女贞(Ligustrum lucidum Ait.)转录组分析结果,利用cDNA末端快速扩增(RACE)技术从叶片中克隆到1个编码糖基转移酶的基因,命名为LlUGT。该基因cDNA全长1621 bp,由47 bp的5′非翻译区、1473 bp的开放阅读框(ORF)和101 bp的3′非翻译区组成,其ORF编码1个含490个氨基酸残基的蛋白(LlUGT),该蛋白的理论相对分子质量和理论等电点分别为55365.68和pI 6.26。生物信息学分析结果表明:LlUGT的氨基酸序列中含有1段糖基转移酶高度保守的区域,即PSPG盒。LlUGT的二级结构含有α-螺旋(41.02%)、β-折叠(10.41%)和无规卷曲(48.57%),三级结构中由肽链折叠形成2个正对的α/β/α类Rossmann折叠区域,并且二者之间夹着1个底物结合口袋。氨基酸序列比对和系统发育分析结果显示:LlUGT与拟南芥(Arabidopsis thaliana(Linn.)Heynh.)UGT74F2的亲缘关系最近,氨基酸序列一致性为47%,且二者具有相同功能的氨基酸残基,故推测LlUGT与UGT74F2功能相似。LlUGT与水杨酸的分子对接实验结果显示:LlUGT中的His51一方面与水杨酸羧基形成氢键,另一方面与Asp143形成氢键,这与UGT74F2的对接结果相同,故推测LlUGT与UGT74F2相同,也是专一催化水杨酸生成糖酯的酶。此外,通过基因工程技术成功在大肠杆菌体内获得LlUGT蛋白。综上所述,本研究首次从女贞叶片中克隆到1个编码糖基转移酶的基因,经理论预测其编码蛋白是1种催化水杨酸生成糖酯的酶。

Based on the results of previous transcriptome analysis for Ligustrum lucidum Ait.,a gene encoding glycosyltransferase was cloned from leaves by using the technology of rapid amplification of cDNA ends(RACE)and named LlUGT.The full length of cDNA of this gene is 1621 bp,it consists of 47 bp 5′-untranslated region,1473 bp open reading frame(ORF)and 101 bp 3′-untranslated region,and the ORF encodes a protein(LlUGT)containing 490 amino acid residues with the theoretical relative molecular mass and theoretical isoelectric point of 55365.68 and pI 6.26,respectively.The bioinformatics analysis results show that the amino acid sequence of LlUTG contains a highly conserved domain of glycosyltransferase,which is PSPG box.The secondary structure of LlUGT containsα-helix(41.02%),β-sheet(10.41%)and random coil(48.57%),and in the tertiary structure,two face-to-faceα/β/αRossmann folded regions are formed by folding peptide chains,with a substrate binding pocket sandwiched between them.Alignment of amino acid sequences and phylogenetic analysis results show that LlUGT has the closest genetic relationship with UGT74F2 from Arabidopsis thaliana(Linn.)Heynh.,the identity of their amino acid sequence is 47%,and they have the amino acid residues with the same functions,so it is speculated that the function of LlUGT is similar to UGT74F2.The result of molecular docking experiment of LlUGT and salicylic acid shows that His51 of LlUGT forms hydrogen bonds with the carboxyl group of salicylic acid on one hand,and with Asp143 on the other hand,which consistent with the molecular docking result of UGT74F2,therefore,it is speculated that LlUGT is also an enzyme that specifically catalyzes the formation of sugar esters from salicylic acid,like UGT74F2.In addition,LlUGT is successfully obtained in E.coli by using genetic engineering technology.In conclusion,a gene encoding glycosyltransferase is cloned from leaves of L.lucidum for the first time in this study,and the encoded protein is theoretically predicted as an enzyme,which can catalyze salicylic acid to generate sugar esters.