拟南芥<em>CPK10</em>双元TAP载体的构建及原生质体表达

doi:10.7668/hbnxb.2015.04.008

华北农学报 ›› 2015, Vol. 30 ›› Issue (4): 43-46. doi: 10.7668/hbnxb.2015.04.008

拟南芥CPK10双元TAP载体的构建及原生质体表达

梁芸, 魏凤菊

河北农业大学生命科学学院, 河北省植物生理与分子病理学重点实验室, 河北保定 071001

收稿日期:2015-04-22 出版日期:2015-08-28
通讯作者: 魏凤菊(1979-),女,河北衡水人,讲师,博士,主要从事植物抵抗逆境分子机制研究。
作者简介:梁芸(1990-),女,河北平山人,在读硕士,主要从事植物逆境研究。
基金资助:
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)

Construction of Binary Vector for TAP and Protoplast Expression of CPK10 in Arabidopsis

LIANG Yun, WEI Feng-ju

Hebei Province Key Laboratory of Plant Physiology and Molecular Pathology, College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China

Received:2015-04-22 Published:2015-08-28

摘要/Abstract

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。

关键词: TAP, 双元表达载体, CPK10, 原生质体转化

Abstract: 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 recombinant plasmid pCM1307-3FLAG-3HA-CPK10 was transformed into Arabidopsis wild-type protoplast mediated with PEG,about 10 hours expression,the CPK10 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 CPK10 interaction proteins through tandem affinity purification technique.

Key words: TAP, Binary expression vector, CPK10, Protoplast transformation

中图分类号:

梁芸, 魏凤菊. 拟南芥CPK10双元TAP载体的构建及原生质体表达[J]. 华北农学报, 2015, 30(4): 43-46. doi: 10.7668/hbnxb.2015.04.008.

LIANG Yun, WEI Feng-ju. Construction of Binary Vector for TAP and Protoplast Expression of CPK10 in Arabidopsis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(4): 43-46. doi: 10.7668/hbnxb.2015.04.008.

http://www.hbnxb.net/CN/Y2015/V30/I4/43

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拟南芥CPK10双元TAP载体的构建及原生质体表达

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拟南芥CPK10双元TAP载体的构建及原生质体表达

Construction of Binary Vector for TAP and Protoplast Expression of CPK10 in Arabidopsis

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