鸢尾糖基转移酶编码基因ItUGT349和ItUGT419克隆及特性研究  

Genes clone and features analysis of glycosyltransferase genes ItUGT349 and ItUGT419 from Iris tectorum

作　　者：张李兵 李静[1] 陶无恙 黄梓璐 陈嘉杰 段礼新 季爱加 ZHANG Li-bing;LI Jing;TAO Wu-yang;HUANG Zi-lu;CHEN Jia-jie;DUAN Li-xin;JI Ai-jia(Guangdong Provincial Key Laboratory of Translational Cancer Research of Chinese Medicines,Joint International Research Laboratory of Translational Cancer Research of Chinese Medicines,International Institute for Translational Chinese Medicine,School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510000,China)

机构地区：[1]广州中医药大学、中药学院国际中医药转化医学研究所,教育部中医药防治肿瘤转化医学研究国际合作联合实验室,广东省中医药防治肿瘤转化药学研究重点实验室,广东广州510000

出　　处：《中草药》2023年第1期254-261,共8页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金青年基金项目(82003895);广东省基础与应用基础研究基金项目(2019A1515110594,2020A1515010926);广东省级大学生创新训练项目(S202110572094,S202110572055)。

摘　　要：目的 克隆获得鸢尾Iris tectorum糖基转移酶基因It UGT349和ItUGT419,并对其进行生物信息学分析、基因差异表达检测和蛋白原核表达等特性分析。方法 以鸢尾转录组中筛选到的It UGTs基因全长开放阅读框(openreadingframe,ORF)设计特异性引物,进行PCR扩增,经测序后获得基因序列并进行生物信息学分析;通过荧光定量PCR(real-time PCR,q RT-PCR)进行基因差异表达检测;最后构建pET-32a(+)原核表达载体在大肠杆菌中表达蛋白。结果 PCR扩增ItUGT349和It UGT419的ORF长度分别为1461、1488 bp,编码蛋白相对分子质量大小分别为53 830、54 910。荧光定量PCR显示,It UGT349的表达量在叶片中最高,而It UGT419在花器官中表达最高。进化树表明,ItUGT419与三萜类糖基转移酶聚类在一起,ItUGT349与三萜、黄酮和木质素等多种类型的糖基转移酶聚类到一支。ItUGT349和ItUGT419在大肠杆菌中均成功的表达出可溶性蛋白。结论 通过It UGT349和ItUGT419基因全长ORF克隆,并对其进行序列分析,基因差异表达检测及原核表达等研究,为后续进一步鉴定其催化功能奠定基础。Objective To clone the glycosyltransferase genes ItUGT349 and ItUGT419 from Iris tectorum and conduct bioinformatics analysis, gene differential expression analysis and protein prokaryotic expression analysis. Methods Gene-specific primers were designed based on the open reading frame(ORF) of ItUGTs, which were screened from the transcriptome data;And the gene products were obtained by PCR amplification. After sequencing identification,bioinformatics analysis was performed. Moreover, the different expression of both genes among various tissues were detected by real-time qPCR. Finally, the target gene was constructed into the prokaryotic expression vector pET-32a(+) and the soluble proteins were obtained in Escherichia coli. Results The length of open reading frame(ORF) of ItUGT349 and ItUGT419were 1461 bp and 1488 bp, respectively;And the molecular weight of the encoding proteins were 53 830 and 54 910. The fluorescence quantitative PCR results showed that the expression level of ItUGT349 was highest in leaves, while ItUGT419was highly expressed in flowers. The phylogenetic tree showed that ItUGT419 was clustered with the UGTs, which were related to triterpene biosynthesis;And ItUGT349 was clustered with the UGTs, which were involved in the biosynthesis of triterpene, flavonoids and lignin. Finally, ItUGT349 and ItUGT419 were successfully expressed in E. coli. Conclusion The full-length ORF cloning, sequences analysis, differential gene expression detection and prokaryotic expression of ItUGT349and ItUGT419 lay a foundation for the subsequent identification of their catalytic function in I. tectorum.

关 键 词：鸢尾 糖基转移酶 基因克隆 生物信息学分析 原核表达 QRT-PCR 

分 类 号：R286.12[医药卫生—中药学]