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利用巢式PCR技术分离获得CaTIP1-1基因上游1749 bp的片段。利用PlantCARE软件预测发现该序列含有启动子典型的基本元件TATA-box、CAAT-box及非生物胁迫相关的元件和光应答元件。以PBI121载体为基础,构建CaTIP1-1启动子驱动下绿色荧光蛋白GFP报告基因植物表达载体。利用叶盘法转化烟草获得转基因植株,通过荧光显微镜能够检测到T1代烟草悬浮细胞系能够稳定表达荧光。结果表明该启动子具有驱动绿色荧光蛋白表达的活性。该研究为今后CaTIP1-1启动子在植物基因工程育种中的应用奠定基础。
The 1749 bp fragment upstream of CaTIP1-1 gene was isolated by nested PCR. PlantCARE software was used to predict that this sequence contains TATA-box, CAAT-box and non-biotic stress-related elements and photo-response elements, which are typical promoter elements. Based on the PBI121 vector, a green fluorescent protein GFP reporter gene expression vector driven by CaTIP1-1 promoter was constructed. Transgenic tobacco plants were obtained by leaf disc transformation. Fluorescence microscopy confirmed the stable expression of T1 in tobacco suspension cell lines. The results show that the promoter has the activity of driving green fluorescent protein expression. This study laid the foundation for future application of CaTIP1-1 promoter in plant genetic engineering breeding.