草莓Fra a基因的克隆与原核表达  

Cloning and Characterization of Fra a Genes from Strawberry (Fragaria × ananassa)

作　　者：李瑞玲[1] 叶云天 江雷雨[1] 刘勇强[1] 叶宇芸 李亚丽[1] 肖婕[1] 李欣[1] 汤浩茹[1] Li Ruiling;Ye Yuntian;Jiang Leiyu;Liu Yongqiang;Ye Yuyun;Li Yali;Xiao Jie;Li Xin;Tang Haoru(College of Horticulture, Sichuan Agricultural University, Chengdu, 611130)

机构地区：[1]四川农业大学园艺学院,成都611130

出　　处：《分子植物育种》2018年第11期3487-3494,共8页Molecular Plant Breeding

基　　金：四川农业大学园艺学院资助

摘　　要：草莓Fra a是一类过敏原蛋白。为研究Fra a蛋白致敏机理,本研究根据已知的栽培草莓(Fragaria×ananassa)Fra a序列设计引物,从"红颜"草莓中克隆得到三个Fra a基因序列,其ORF长度分别为483 bp、483 bp和480 bp,各自编码160个、160个和159个氨基酸,分别命名为Fa Fra a 1、Fa Fra a 2和Fa Fra a 3。生物信息学预测表明其均具有bet v 1家族保守结构域,预测蛋白均无跨膜结构域和信号肽。Fa Fra a 1与桃的氨基酸相似度为81%,李、杏为80%;Fa Fra a 2与月季的氨基酸相似度为82%,苹果、樱桃为81%;Fa Fra a 3与月季的氨基酸相似度为92%,苹果为83%。Fra a与蔷薇科植物的进化关系更近。构建原核表达载体pET32afraa1、pET32a-fraa2和pET32a-fraa3,转化到大肠杆菌BL-21(DE3)p Lys S菌株并诱导融合蛋白表达,结果表明目的蛋白Fra a 1、Fra a 3在原核表达系统中能顺利表达,Fra a 1在上清、沉淀中均有大量分布,Fra a 3主要存在于沉淀中。未得到Fra a 2目的蛋白。本研究为进一步了解Fra a的功能和制备抗体提供了理论依据。Fra a is one of the allergen proteins. For further study of the allergenicity of Fra a proteins and gene functions, three gene sequences were gained, named FaFra a 1, FaFra a 2 and FaFra a 3,whose ORF are separately 483 bp, 483 bp and 480 bp, encoding 160, 160 and 159 amino acids By bioinformation analysis, Fra a had the characteristic conservative domains of the bet v 1 family, and had no trans-membrane domain or signal peptide.FaFra a 1 had 81% ident with Prunus persica, and 80% with Prunus domestica and Prunus armeniaca. FaFra a 2 had 82% ident with Rosahybrid cultivar, and 81% with Malus domestica and Prunus avium. FaFra a 3 had 92%ident with Rosahybrid cultivar, and 83% with Malus domestica. Phylogenetic analysis showed that Fra a proteins were clustered together with plants in rose family. Constructing prokaryotic expression vector pET32 a-fraa1,pET32 a-fraa2 and pET32 a-fraa3, transforming into Escherichia coli BL-21（DE3） p Lys S to induce fusion protein.The result showed that the target protein Fra a 1 and Fra a 3 can be well expressed in the prokaryotic expression system. Fra a 1 can be found in supernatant and precipitate a lot, however, Fra a 3 can be found little in supernatant,but precipitate a lot. Fra a 2 was not gained. This study can further be applied to preparation of antibodies and the study of gene functions.

关 键 词：草莓 过敏原蛋白 克隆 原核表达 黄酮类化合物 

分 类 号：Q943.2[生物学—植物学] S668.4[农业科学—果树学]