| 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-TVector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ⅠKlenow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pBAF。 |
| 关键词: 胡萝卜 抗冻蛋白基因 分子生物学 |
| DOI： |
| 分类号:F061.5 |
| 基金项目:陕西省科技攻关项目(91K12-G19(11)) |
|
| Cloning of a carrot gene encoding antifreeze protein and construction of its plant expression vector |
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JIA Zhi kuan,DAI Jun,JIA Zhi kuan,DAI Jun,JIA Zhi kuan,DAI Jun
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| 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 EcoRⅠand became linear.Its ends were filled with DNA PolymeraseⅠKlenow fragment.Then it was digested with XbaⅠand a designed fragment (afp) with a cohesive end and a blunt end was released.Plant 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. |
| Key words: carrot antifreeze protein gene molecular biology |
