Analysis of a Transcription Factor Using Transient Assay in Arabidopsis Protoplasts
Analysis of a Transcription Factor Using Transient Assay in Arabidopsis Protoplasts
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DOI：
10.1007/978-1-61779-154-3_6
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Koizumi, Nozomu
中科院分区：
文献类型：
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作者：
Iwata, Yuji;Lee, Mi-Hyun;Koizumi, Nozomu
转录因子对基因表达的调控是细胞过程基本上所有方面的基本机制。含有由感兴趣的启动子驱动的报告基因的报告质粒和表达转录因子的效应质粒的瞬时表达测定已经成为分析转录因子的有力工具。在这里，我们提出了一个协议，聚乙二醇（PEG）介导的转化拟南芥原生质体。它详细说明了从拟南芥悬浮培养细胞或土壤生长的拟南芥植物的叶片制备原生质体，以及随后用报告和效应质粒进行PEG介导的转化。该方案可以在24小时内完成从原生质体制备到报告基因测定。作为一个实例，显示了膜结合转录因子AtbZIP60及其靶BiP3启动子的分析。
Regulation of gene expression by transcription factors is a fundamental mechanism in essentially all aspects of cellular processes. Transient expression assay of a reporter plasmid containing a reporter gene driven by a promoter of interest and an effector plasmid expressing a transcription factor has been a powerful tool for analyzing transcription factors. Here we present a protocol for polyethylene glycol (PEG)-mediated transformation of Arabidopsis protoplasts. It details preparation of protoplasts from Arabidopsis suspension cultured cells or leaves of soil-grown Arabidopsis plants and subsequent PEG-mediated transformation with reporter and effector plasmids. This protocol can be completed within 24 h from protoplast preparation to reporter assay. As an example, analysis of the membrane-bound transcription factor AtbZIP60 and its target BiP3 promoter is shown.