黑莓糖基转移酶基因UGT78H2的分离鉴定及与类黄酮化合物的分子对接

Characterization of the Glycosyltransferase Gene UGT78H2 from Blackberry and Docking with Flavonoid Molecules

糖基化修饰对于维持植物小分子化合物的多样性具有重要的作用。糖基转移酶是催化该反应发生的主体,但其底物受体和供体特异识别机制仍未清楚。本研究从黑莓(Rubus spp.)材料中获得了一个糖基转移酶全长编码基因,并提交国际糖基转移酶命名委员会命名为UGT78H2(Gen Bank登录号:KM061379)。该基因编码区全长1 380 bp,共编码459个氨基酸。编码蛋白的预测分子量为50.6 k D,等电点为5.88。生物信息学分析表明该酶具有植物糖基转移酶超家族I成员具有的植物次生代谢产物糖基转移酶保守区;系统发育树分析,UGT78H2属拟南芥糖基转移酶F类群,可能参与类黄酮化合物糖基化。氨基酸水平与月季Rh UF3GT1(UGT78H1)最相似,一致性为88%。UGT78H2在黑莓幼果期表达量最高,随着果实成熟,表达量迅速下降。以蒺藜苜蓿UGT78G1(PDB:3HBF)蛋白晶体结构为模板,同源模建了UGT78H2的结构,以其鉴定的受体催化活性中心与常见的11种类黄酮小分子进行分子对接。结果表明,UGT78H2最有可能催化的三种类黄酮化合物依次为黄酮醇槲皮素,柚皮素和黄烷醇表儿茶素。

Glycosylation plays a pivotal role in determining the diversities of plant small molecules.Glycosyltransferases are ubiquitous proteins involved in such reactions. However,the exact mechanisms of sugar donor or acceptor substrate recognition are still to be elusive. We reported the isolation of a new glycosyltransferase gene from blackberry plants（ Rubus spp.）,nominated as UGT78H2（ Gen Bank Accession No. ： KM061379） by the international UDP Glucosyltransferase Nomenclature Committee. The full length of the gene contained an open reading frame of 1 380 nucleotides,encoding a 50. 6 k D polypeptides of 459 amino acids. By further analysis,the deduced enzyme was a typical family-1 glucosyltransferase with the plant secondary product glucosyltransferase signature motif in the C terminal. Phylogenetic trees revealed the clustering of UGT78H2 in the F node together with other Arabidopsis thaliana GTs,inferring the possible catalysis activity toward flavonoid compounds. Amino acids of UGT78H2 shared most,but only 88% identity to the Rh UF3GT（ UGT78H1） in Rose hybrid when aligned to other glycosyltransferases. Most abundant transcripts of the gene were observed in the immature blackberry fruits,a stage when little anthocyanins were produced.Additionally,transcription levels decreased dramatically with the maturation of fruits. We used the Medicagotruncatula UGT78G1（ PDB： 3HBF） as a template to homologically model the protein of UGT78H2 that was subsequently docked with 12 common flavonoid molecules. UGT78H2 was predicted to be a glucosyltransferase having maximum binding affinity towards quercetin,tangeritin and epicatechin.