【摘要】 以胡萝卜品种 Autumn King的幼苗为材料 ,用 CTAB法提取其基因组 DNA,以 PCR ( Polym eraseChain Reaction)的方法在体外扩增出胡萝卜抗冻蛋白基因 ( afp) ,以 p UCm - T Vector为载体构建成胡萝卜 afp的克隆载体 p TAF,用 Eco R 消化重组质粒 p TAF使其线性化 ,再用 DNA聚合酶 Klenow大片段补平末端 ,然后用Xba 消化 ,获得一末端粘 ,一末端平的目的片段 ( afp)。植物表达载体 p BI12 1用 X ba 和 Sma 双酶切 ,获得一末端粘 ,一末端平的线性质粒。将目的片段与线性质粒在 T4DNA连接酶的作用下进行定向连接 ,构建成胡萝卜 afp的植物表达载体 p BAF更多还原

【Abstract】 Genomic DNA in the seedlings of carrot cultivar Autumn King was extracted with CTAB method.The carrot antifreeze protein gene (afp) was amplified by PCR(Polymerase Chain Reaction).Cloning vector pTAF of carrot afp was constructed with pUCm T Vector.PTAF was digested with Eco RⅠ and became linear.Its ends were filled with DNA Polymerase Ⅰ Klenow fragment.Then it was digested with Xba Ⅰ and a designed fragment (afp) with a cohesive end and a blunt end was released.Plant expression vector pBI121 was digested with Xba Ⅰ and Sma Ⅰand a linear plasmid with a cohesive end and a blunt end was obtained.The linear plasmid and the designed fragment (afp) were directively ligated with T4 DNA ligase,and the plant expression vector of carrot afp was constructed.更多还原