NtDREB2-PDI融合蛋白的纯化及与DRE元件结合能力的检测  

Purification of NtDREB2-PDI Fusion Protein and Function Analysis of Binding to DRE Element

作　　者：冯锋[1] 刘卫群[1] 

机构地区：[1]河南农业大学生命科学学院,郑州450002

出　　处：《植物生理学通讯》2008年第2期329-333,共5页Plant Physiology Communications

基　　金：河南省科技攻关项目(0624050012)

摘　　要：将从普通烟草(Nicotiana tobaccum)‘K326’中克隆得到的转录因子基因NtDREB2,采用PCR去除NtDREB2基因的终止密码子,酶切后构建入PDI/pET28a融合表达载体。取测序正确的重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导,SDS-PAGE检测表达后,用Ni-NTA纯化融合蛋白,获得了高效表达的可溶性目的蛋白。凝胶滞留(EMSA,electrophoresis mobility shift assay)实验表明融合蛋白具有结合DRE(dehydration-responsive element)元件的能力。The transcription factor gene NtDREB2 is cloned from tobacco （Nicotiana tobaccum）‘K326＇. The stop codon of the NtDREB2 gene is deleted with PCR, and we construct the gene into the PDI/pET28a fusion expression vector after enzyme digestion. The E. coil BL21 （DE3） is transformed with the recombinant plasrnid whose sequence is verified, then induced with IPTG. After SDS-PAGE detection of the expression we acquire the high-expressing soluble protein with the Ni-NTA purification method. EMSA experiment suggests that the fusion protein is capable of binding to the DRE element.

关 键 词：NtDREB2 蛋白质二硫键异构酶 融合蛋白 诱导表达 纯化 凝胶滞留 

分 类 号：Q946[生物学—植物学]