岩白菜糖基转移酶基因BpUGTs1的克隆与表达分析  

Cloning and Expression Analysis of Glucosyltransferase Gene BpUGTs1 in Bergenia purpurascens

作　　者：李沛欣 王仕玉[2] 郭凤根[1] 李娟[1] Li Peixin;Wang Shiyu;Guo Fenggen;Li Juan(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming,650201;College of Horticulture and Landscape,Yunnan Agricultural University,Kunming,650201)

机构地区：[1]云南农业大学农学与生物技术学院,昆明650201 [2]云南农业大学园林园艺学院,昆明650201

出　　处：《分子植物育种》2022年第20期6696-6704,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(81360610)资助。

摘　　要：根据岩白菜转录组数据库中的岩白菜糖基转移酶候选基因Unigene0054989的全长序列设计引物后克隆获得BpUGTs1,并用生物信息学方法对其进行分析;采用RT-qPCR技术分析了该基因在岩白菜各组织中的表达情况;构建pBIl21-BpUGTs1-eGFP植物表达载体并转化农杆菌GV3101,使用农杆菌介导的烟草瞬时表达技术预测和观察BpUGTs1的亚细胞位置。结果表明:所获序列全长1606 bp,可编码498个氨基酸,相对分子质量55.57 kD,属于不稳定蛋白;该序列与没食子酸葡萄糖基转移酶基因相似度高达80%以上,与茶树和阿月浑子的同源序列亲缘关系最近;亚细胞定位BpUGTs1在细胞质上;BpUGTs1在根状茎和胚性愈伤组织表达量最高。综合生物信息学分析结果、亚细胞定位和基因表达量,BpUGTs1可能是在根状茎中合成岩白菜素的关键酶基因。研究结果为该基因的后续遗传转化,基因表达和生物学功能分析提供理论基础。According to the full-length sequence of glucosyltransferase gene(Unigene0054989)from transcriptome data of Bergenia purpurascens,the primers were designed and the gene BpUGTs1 was cloned and analyzed by bioinformatics.The RT-qPCR method was used to analyze the expression quantities of this gene in different tissues of B.purpurascens.Recombinant pBIl21-BpUGTs1-eGFP plant expression plasmid were established and transformed into Agrobacterium strain GV3101 for tobacco transformation.Subcellular localization of BpUGTs1was predicted and observed by bioinformatics analysis and Agrobacterium-mediated transient expression into tobacco leaf,respectively.The results showed that the total length of BpUGTs1 was 1606 bp and encoded 498 amino acids with 55.57 kD of relative molecular weight,belonging to the unstable protein.Phylogenetic tree analysis showed that the sequence was similar to gallate 1-beta-glucosyltransferase gene above 80%and had the closest relationship with those of Camellia sinensis and Pistacia vera.Subcellular localization revealed that the BpUGTs1was located in the cytoplasm.Fluorescent quantitative PCR analysis displayed the BpUGTs1 gene expressed the highest levels in the rhizome and callus.Based on the results of bioinformatics analysis,subcellular location and gene expression,BpUGTs1 may be the key enzyme gene for the synthesis of bergenin in rhizomes.The results provide a theoretical basis for the subsequent genetic transformation,gene expression and biological function analysis of this gene.

关 键 词：岩白菜 糖基转移酶 基因克隆 亚细胞定位 RT-QPCR 

分 类 号：S567.239[农业科学—中草药栽培]