蒙古沙冬青AmDREB2.1表达载体构建及其结合活性鉴定

Constructing of Expressing Vector and Identifying of Binding Activity of AmDREB2.1 in Ammopiptanthus mongolicus

以蒙古沙冬青为试材,以AmDREB 2.1为研究对象,采用构建植物表达载体和叶盘法进行了AmDREB 2.1转烟草研究,采用Gateway和酵母单杂交技术研究了AmDREB 2.1转录因子的结合活性。结果表明:以BamH I和Sac I酶切位点成功构建了pPZP212-AmDREB 2.1表达载体,通过叶盘法获得了T0转AmDREB 2.1烟草株系。成功构建了pDEST22-AmDREB 2.1的酵母单杂交载体,分别转化YH4271-mDRE和YH4271-wDRE 2种酵母菌株,在Trp^-His^-Ade^-三缺培养基(3-AT浓度为0、20mmol·L^(-1))上筛选获得了阳性克隆,检测其β-半乳糖苷酶活性为阳性,显示AmDREB 2.1具有转录因子的结合活性。

Ammopiptanthus mongolicus was used as material,the AmDREB 2.1 transformation to tobacco was investigated via expression vector construction and leaf disc method,and the binding activity of AmDREB2.1was studied with Gateway technique and the yeast one-hybrid assay.The results showed that pPZP212-AmDREB2.1expression vector was constructed with BamH I and Sac I,and then successfully transformed to the tobacco.The pDEST22-AmDREB 2.1,the yeast one-hybrid vector,was constructed,and then were converted into two yeast strains YH4271-mDRE and YH4271-wDRE,respectively.Positive clones were obtained with the ‘Trp^-His^-Ade^-＇selective media（the concentration of 3-AT was 0mmol·L-1 and 20mmol·L-1）,and the activity of β-galactosidase was detected and it indicated that AmDREB 2.1could bind to DREcis-element.