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拟南芥 CPK10双元 TAP 载体的构建及原生质体表达 被引量：2

Construction of Binary Vector for TAP and Protoplast Expression of CPK10 in Arabidopsis
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摘要 为了研究拟南芥CPK10发挥功能的分子机理,通过PCR扩增克隆了CPK10基因,将该基因连接到带有FLAG和HA标签的双元串联亲和层析载体上,构建成p CM1307-3FLAG-3HA-CPK10植物表达载体,进而通过PEG介导转化拟南芥野生型原生质体,表达约10 h,通过Western Blotting检测融合有FLAG和HA标签的CPK10蛋白的表达情况,结果显示,可以分别利用FLAG抗体和HA抗体特异检测到CPK10蛋白的条带。融合蛋白的成功表达,为进一步通过串联亲和纯化技术(TAP)筛选CPK10的互作蛋白奠定基础。 In order to study the molecular mechanism of Arabidopsis CPK10,the gene was amplified by PCR, and constructed into the binary vector with FLAG and HA tags for tandem affinity purification（TAP）.Then the re-combinant plasmid pCM1 307-3FLAG-3HA-CPK10 was transformed into Arabidopsis wild-type protoplast mediated with PEG,about 1 0 hours expression,the CPK1 0 fusion protein with FLAG and HA tags was detected by Western Blotting.Immunoblot analysis performed with FLAG and HA antibodies showed a distinct cross-reaction with fusion protein at expected molecular weight.The successful expression of fusion protein in Arabidopsis protoplast laid the foundation for screening CPK1 0 interaction proteins through tandem affinity purification technique.
作者 梁芸 魏凤菊
机构地区 河北农业大学生命科学学院
出处 《华北农学报》 CSCD 北大核心 2015年第4期43-46,共4页 Acta Agriculturae Boreali-Sinica
基金 国家自然科学基金专项基金项目(31040052) 国家自然科学基金青年基金项目(31101022) 中国农业大学开放课题(SKLPPBKF1504)
关键词 TAP 双元表达载体 CPK10 原生质体转化 TAP Binary expression vector CPK10 Protoplast transformation
分类号 Q78 [生物学—分子生物学]
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参考文献18

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二级参考文献36

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华北农学报
2015年 第4期

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