天山雪莲UDP葡萄糖-类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析

Identification and characterization of a flavonoid-3-O-glucosyltransferas gene from Saussurea involucrata

摘要:

UDP葡萄糖-类黄酮-3-O-葡萄糖基转移酶 (Flavonoid-3-O-glucosyltransferas，3GT) 是植物重要的次生代谢产物生物合成途径中的关键酶。文中采用现代生物信息手段，经3GT的同源比对后设计基因特异引物，运用RT-PCR及RACE技术从天山雪莲Saussurea involucrata Kar. et Kir.叶片中克隆得到3GT基因的全长序列 (GenBank Accession No. JN092127)。3GT基因的cDNA全长序列含有1个1 548 bp的开放阅读框 (ORF)，编码516个氨基酸，该基因推断的蛋白与草莓GT6蛋白的相似性为91％，与毛杨梅3GT的相似性为89％；经序氨基酸序列比对，推断的天山雪莲3GT具有糖基转移酶基因家族特有的结构域PSPG-box。半定量PCR的结果显示，天山雪莲3GT基因在叶及愈伤组织中表达量最高，在根中有少量表达，茎中不表达。将该基因构建到含有35S启动子的植物表达载体上，利用农杆菌介导的遗传转化法进行同源转化，将筛选到的含有转3GT基因的愈伤组织进行悬浮培养，并用紫外分光光度法测量其黄酮含量是非转基因愈伤组织总黄酮平均值的2.06倍。

Abstract:

The flavonoid-3-O-glucosyltransferas (3GT) is one of the most important enzymes for biosynthesis of plant secondary metabolites. In this paper, the homology analysis and gene special primers design were used. With the methods of mordern molecular biology, the full-length gene of 3GT (GenBank Accession No. JN092127) which was cloned from Saussurea involucrata Kar. et Kir by RT-PCR and RACE. The cDNA sequence of 3GT consisted of 1548 bp open reading frame (ORF) encoding 516 amino acid, the deduced 3GT protein shared 91% and 89% identities with that of Fragaria x ananassa GT6, Manihot esculenta anthocyanidin 3-O-glucosyltransferase. Homology analysis showed that deduced 3GT protein has a glycosyltransferase signature domain PSPG-box. The transcripts of 3GT members were found mainly in leaves and callus. 3GT gene of S. involucrata was under the control of the cauliflower mosaic virus (CaMV) 35S promoter, homologous transformation used an Agrobacterium rihizogenes-mediated transformation system. The results on UV spectrophotometry showed S. involucrata callus after suspension culture that an average of total flavonoids on transgenic callus was 2.06 times higher than non-transgenic callus.