毛白杨NBS型基因PtDRG01原核表达研究  

Prokaryotic Expression Analysis of A NBS-type PtDRG01 Gene Isolated from Populus tomentosa Carr.

作　　者：李琰[1] 张谦[1] 李海霞[1] 刘婷婷[1] 安新民[1] 张志毅[1] 

机构地区：[1]北京林业大学林木花卉遗传与育种教育部重点实验室,北京100083

出　　处：《西北植物学报》2008年第5期882-888,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：教育部博士点基金项目(20070022003)

摘　　要：为了分析毛白杨NBS型抗病基因PtDRG01(EF157840)编码蛋白的特性,研究构建了该基因的原核表达载体pGEX-Pt01,并导入大肠杆菌XA90,经IPTG诱导表达后,SDS-PAGE电泳分析证明,该特异蛋白的分子量约为79kD。对原核表达体系以及融合蛋白的表达特性进行优化与分析表明:最佳诱导表达条件为1mmol/L IPTG在37℃下诱导4h,所表达的融合蛋白为胞内分泌的可溶性蛋白。利用谷胱苷肽-琼脂糖亲和层析柱纯化获得了电泳纯级的目标蛋白,为PtDRG01基因编码蛋白的功能鉴定研究奠定了基础。In order to investigate the protein features of a NBS gene （PtDRG01, EF157840） isolated from Populus tomentosa Carr. ,the full-length open reading frame was fused into a prokaryotic expression vector pGEX-KG. PCR analysis and double endonucleases digestion showed that the recombinant vector was successfully constructed and transferred into an expression host E. coli strain XA90. It was indicated by SDS- PAGE analysis that IPTG treatment successfully induced the expression of a fusion protein about 79 kD, which was consistent with the predicted value. In addition,The prokaryotic expression system was also optimized. The result suggested that 1 mmol/L IPTG treatment for 4 h at 37℃ was most effective,and the product was predominately soluble and not extra-cellular secreting. Moreover, the fusion protein was purified with the affinity chromatography column of glutathione sepharose 4B. This work lays the foundation for further study on biological functions of PtDRG01 gene.

关 键 词：毛白杨 NBS型基因 原核表达 蛋白纯化 

分 类 号：Q789[生物学—分子生物学]