CAJ | 학술논문

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 pCM1307-3FLAG-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。
위료연구의남개 CPK10발휘공능적분자궤리，통과 PCR 확증극륭료 CPK10기인，장해기인련접도대유FLAG 화 HA 표첨적쌍원천련친화층석재체상，구건성 pCM1307-3FLAG-3HA-CPK10식물표체재체，진이통과 PEG 개도전화의남개야생형원생질체，표체약10 h，통과 Western Blotting 검측융합유 FLAG 화 HA 표첨적 CPK10단백적표체정황，결과현시，가이분별이용 FLAG 항체화 HA 항체특이검측도 CPK10단백적조대。융합단백적성공표체，위진일보통과천련친화순화기술（TAP）사선 CPK10적호작단백전정기출。
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.