拟南芥CBF4基因启动子缺失载体的构建及转化

Subcloning and Transformation of the Deletion Vectors of CBF4 Promoter from Arabidopsis thaliana

利用PCR方法,从拟南芥基因组中克隆脱水响应因子CBF4基因启动子全序列4A(1 260 bp)及两段不同长度5′缺失片段4B(565 bp)和4C(470 bp),通过亚克隆构建含有该系列启动子的可溶性绿色荧光蛋白基因(smGFP)的植物表达载体p1301-4A-GFP、p1301-4B-GFP、p1301-4C-GFP,用农杆菌介导烟草叶盘法分别导入烟草。20%PEG6000模拟干旱诱导转基因植株,检测转基因烟草转入不同启动子片段烟草植株诱导前后叶片GFP的表达强度。在倒置荧光显微镜和384 nm紫外灯照射下检测到的全片段4A与5′缺失片段4B和4C转化植株均显荧光,且随启动子长度的变短,荧光强度逐渐增强。基本确定了启动子的核心区域为4C(470 bp)。

In this study, the promoter sequence of dehydration response factor CBF4 4A（ 1 206 bp）, two 5＇ deletion fragments 4B（565 bp） and 4C（470 bp） from the Arabidopsis genome were cloned by PCR. The soluble green fluorescent protein gene （smGFP） was constructed with this series of promoter into vec- tor pl301-GFP by subcloning, then transformed into tobacco via Agrobacterium-mediated by tobacco leaf disc transformation. Transgenic plants were induced with simulated drought, and GFP expression intensity of transgenic tobacco leaves before and after induction was detected. Under the inverted fluorescence mi- croscope and 384 nm irradiation of the UV light to detect the fragments of 4A, 4B and 4C deletion of transformed plants showed that as promoter length became shorter, fluorescence intensity was greater. The core region of promoter was 4C（470 bp）.