烟草DREB-like转录因子的克隆与鉴定  

Cloning and Identification of DREB-like Transcription Factor in Nicotiana benthamiana

作　　者：刘卫群[1] 王永亮[2] 赵同金[3] 周海梦[3] 郭蔼光[1] 

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]河南农业大学农业生物技术系,郑州450002 [3]清华大学生物科学与技术系,北京100084

出　　处：《农业生物技术学报》2006年第3期376-380,共5页Journal of Agricultural Biotechnology

摘　　要：在NCBI网站BLAST得到5个烟草表达序列标签(EST),据此设计引物,从烟草(Nicotianabenthamiana)品种本塞母氏中分离出2条DREB(dehydration-responsiveelementbindingprotein)-like转录因子基因,分别命名为NbDREB1和NbDREB2。与相关AP2/EREBP类转录因子比对结果显示,NbDREB1和NbDREB2都具有典型的AP2/EREBP转录因子家族EREBP亚族A类AP2区特征,并且都能在大肠杆菌(Escherichiacoli)中表达。酵母单杂交结果显示,都不具有激活功能。连接pGADT7反式激活载体进行酵母单杂交结果显示:NbDREB1能与DRE顺式作用元件结合,NbDREB2则不能,表明NbDREB1为抑制型DREB类转录因子。比较NbDREB1和NbDREB2的AP2区发现:两者在第2位和第49位氨基酸处不同,NbDREB1为Y和K,NbDREB2为N和R。Five EST （expressed sequence tag） sequences were obtained in NCBI web BLAST according to the AP2 domain sequence of DREB（dehydration-responsive binding protein）1 a gene from Arabidopsis. Specific primers were designed based on the EST sequences, and two DREB-Iike genes were amplified from tobacco （Nicotiana benthamiana） cDNA by PCR, and named NbDREBI and NbDREB2 respectively and could be expressed in Escherichia coll. Comparing with other dehydration-responsive element binding related protein found that NbDREBI and NbDREB2 possessed typical structural characteristics of A type of EREBP subgroup in AP2/EDREBP transcription factor family. The yeast one-hybrid （expression vector YepGap） showed that the two genes had no active function. Anti-activator yeast one-hybrid （expression vector pGADT7containning GAL4 domain） assay showed that NbDREBI could bind to DRE c/s-element in yeast, but NbDREB2 could not. The difference of AP2 domain between NbDREBI and NbDREB2 was only in the 2nd and 49th amino acid, which was Y and K in NbDREBI and N and R in NbDREB2.

关 键 词：烟草 DREB 克隆 酵母单杂交 反式激活 

分 类 号：S572[农业科学—烟草工业]