摘要
从烟草品种本塞母氏中分离出2条DREB类转录因子基因,分别命名为NbDREB1和NbDREB2.根据测序结果推导出的氨基酸序列分析显示,NbDREB1和NbDREB2都具有典型的AP2/EREBP转录因子家族EREBP亚族A类特征.酵母单杂交结果显示,它们都不具有激活功能.连接pGADT7反式激活载体形成融合基因表达结果显示,NbDREB1能与DRE顺式作用元件结合,NbDREB2则不能.比较NbDREB1和NbDREB2的AP2区,发现两者的第2和49位氨基酸残基不同.对NbDREB2的第2位氨基酸残基N点突变为Y,NbDREB2也显示出与DRE顺式元件结合的活性,表明烟草DREB转录因子的AP2区第2位氨基酸残基Y是识别及结合DRE顺式作用元件必需的氨基酸残基.
Two DREB-like genes were obtained from Nicotiana benthamiana and named NbDREB1 and NbDREB2, respectively. The deduced amino acid sequences from NbDREB 1 and NbDREB2 possessed typical structural characteristics of A-type of EREBP subgroup in AP2/EREBP transcription factor family. The results of yeast one-hybrid showed that the two genes did not activate the transcription of the dual reporter gene in yeast. NbDREB1 and NbDREB2 were constructed into pGADT7 trans-acting vector respectively. NbDREB1 and NrDREB2 genes coding sequence fused to the end of the GAlA activation domain. Yeast in vvvo analysis showed that on a selective medium plate of SD/His^- Ura^- Trp^- ＋ 0.03 mol/L 3-aminotriazole, the growth of yeast cells containing His and LacZ double reporter gene was normal and induced LacZ activity in the transformation of NbDREB1. While neither cell growth nor induced LacZ activity in the transformation of NbDREB2 was observed. These data indicated that NbDREB1 was a kind of inhibiting AP2/EREB transcription factor gene. Comparing the AP2 domain between the NbDREBI and NbDREB2, all of the consensus amino acids were conserved in the NbDREB1 and NbDREB2 except that the 2nd and 49th amino acids were Y （ tyrosine ） and K （ lysine ） in the NbDREB1 and N （ asparagine ） and R （ arginine ） in the NbDREB2 transcription factor. The 2nd amino acid of AP2/EREBP domain of the NbDREB2 was mutated in which N（2nd） residues was replaced by Y, showing the same activity as NbDREB 1. The results suggested that the conserved Y（2nd） residue might be pivatal regarding the specificity of binding to target sequence of ciselements in Nicotiana benthamiana.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第2期111-116,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家高技术研究发展计划(863计划)资助项目(No.2002AA224091)~~
