小立碗藓GFP-CAD1-PNT182融合基因表达载体的构建

发布时间：2018-05-13 20:36

  本文选题：GFP + 高保真酶　； 参考：《西南农业学报》2017年09期

【摘要】：【目的】为利用绿色荧光蛋白基因(GFP)的表达情况优化聚乙二醇(PEG)介导的小立碗藓原生质体的转化体系及检测CAD1基因在细胞中的定位,构建小立碗藓GFP-CAD1-PTN182融合基因表达载体。【方法】用高保真酶获得GFP基因,通过双酶切GFP基因和CAD1-PTN182表达载体,将两片段洗脱回收,然后用T4-DNA连接酶将酶切产物在16℃条件下过夜连接。将连接产物转化到E.coli DH5α感受态细胞中,通过凝胶成像电泳的方法,挑选阳性克隆进行验证,并经过酶切验证和测序。【结果】序列对比相似度达100%,表明GFP-CAD1-PTN182融合基因表达载体构建成功。【结论】该载体的成功构建,为PEG介导小立碗藓原生质体转化体系的优化提供了便捷的检测手段,同时为进一步研究CAD1基因在细胞中的定位提供了理论依据。
[Abstract]:[objective] to optimize the transformation system of PEG- mediated protoplasts and to detect the location of CAD1 gene in cells by using the expression of GFP gene. The expression vector of GFP-CAD1-PTN182 fusion gene was constructed. [methods] GFP gene was obtained by high fidelity enzyme. The two fragments were eluted and recovered by double enzyme digestion of GFP gene and CAD1-PTN182 expression vector. The digested products were linked overnight at 16 鈩，

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