一个潜在催化莱鲍迪D苷合成的新型糖基转移酶  

A Novel Glycosyltransferase Potentially Catalyzing the Synthesis of Rebaudioside D

作　　者：陈红 张绍山 肖婕妤 陈艾萌 陈治宇 王曦旎 吴卫[1] Chen Hong;Zhang Shaoshan;Xiao Jieyu;Chen Aimeng;Chen Zhiyu;Wang Xini;Wu Wei(College of Agronomy,Sichuan Agricultural University,Chengdu,611130)

机构地区：[1]四川农业大学农学院,成都611130

出　　处：《分子植物育种》2020年第9期2872-2877,共6页Molecular Plant Breeding

基　　金：国家自然科学基金(No.31671757);四川农业大学创新训练计划项目(20161062604)共同资助.

摘　　要：尿苷二磷酸糖基转移酶(uridine diphosphate glycosyltransferases,UGTs)催化糖基转移反应,与植物次生代谢密切相关。本研究根据甜叶菊(Stevia rebaudiana)转录组数据库,克隆到一个催化莱鲍迪D苷(rebaudioside D,RD)合成的新型糖基转移酶候选基因,对其开展生物信息学分析。结果表明,该基因开放阅读框长1380 bp,编码459个氨基酸,等电点(pI)预测为5.54,理论分子量约49.66 kD,系统发育分析表明该基因与向日葵中的UGT89A2同源,故将其命名为SrUGT89A2。构建pET28a-SrUGT89A2原核表达载体,并在大肠杆菌(BL21(DE3))中诱导表达得到重组蛋白,HPLC检测表明粗酶液能催化甜叶菊提取液形成一个新的色谱峰,该峰保留时间与莱鲍迪D苷一致。经进一步纯化UGT89A2蛋白,添加不同甜菊糖苷标准品为催化底物,但未鉴定出该蛋白催化的具体糖苷。该潜在催化甜菊糖RD苷合成的新型糖基转移酶基因SrUGT89A2的发现,为RD苷的生物合成和甜菊糖苷的生物途径研究提供新的理论依据。Uridine diphosphate glycosyltransferases(UGTs)catalyze the glycosylation reaction,which are closely related to the plant secondary metabolism.In the present study,a novel putative glycosyltransferase gene catalyzing the biosynthesis of RD was cloned based on the transcriptome data of Stevia rebaudiana.The sequence analysis showed that the ORF of the gene was 1380 bp and encoded 459 amino acids.The isoelectric point(pI)was predicted to be 5.54 and the theoretical molecular weight was 49.66 kD.Phylogenetic analysis showed that the gene was clustered with UGT89A2 which was identified from Helianthus annuus.SrUGT89A2 was expressed in Escherichia coli BL21(DE3)and high-performance liquid chromatography(HPLC)analysis of the reaction products showed that the recombinant fusion protein could catalyze S.rebaudiana extracts to form a new chromatographic peak,which was consistent with rebaudioside D(RD).In addition,in vitro enzyme assay of purifed recombinant protein was conducted using different steviol glycosides as the sugar acceptors,but the specific glycosides were not identified.The discovery of SrUGT89A2,a novel glycosyltransferase gene that potentially catalyzes the synthesis of RD,provides a new theoretical basis for the biosynthesis of RD.

关 键 词：甜叶菊(Stevia rebaudiana) SrUGT89A2 RD苷 基因克隆 功能分析 

分 类 号：Q946.5[生物学—植物学]