胡萝卜抗冻蛋白基因克隆及植物表达载体构建

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 (Polymerase Chain Reaction)的方法在体外扩增出胡萝卜抗冻蛋白基因(afp),以pUCm-T Vector为载体构建成胡萝卜afp的克隆载体pTAF,用EcoRⅠ消化重组质粒pTAF使其线性化,再用DNA聚合酶Ⅰ Klenow大片段补平末端,然后用XbaⅠ消化,获得一末端粘,一末端平的目的片段(afp)。植物表达载体pBI121用XbaⅠ和SmaⅠ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T4 DNA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pBAF。%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 constru cted with pUCm-T Vector.PTAF was digested with EcoRⅠ and became linear.Its ends were filled with DNA Polymerase Ⅰ Klenow fragment.Then it was digested wi th XbaⅠ and a designed fragment (afp) with a cohesive end and a blunt end was released.Plant expression vector pBI121 was digested with XbaⅠ and SmaⅠand 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.