Iwata Yuji, Lee Mi-Hyun, Koizumi Nozomu
Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA.
Methods Mol Biol. 2011;754:107-17. doi: 10.1007/978-1-61779-154-3_6.
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.
转录因子对基因表达的调控是细胞过程几乎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与表达转录因子的效应质粒的瞬时表达分析，一直是分析转录因子的有力工具。在此，我们介绍一种聚乙二醇（PEG）介导的拟南芥原生质体转化方法。该方法详细说明了从拟南芥悬浮培养细胞或土培拟南芥植株的叶片制备原生质体，以及随后用报告质粒和效应质粒进行PEG介导的转化过程。从原生质体制备到报告基因检测，该方法可在24小时内完成。作为一个例子，展示了对膜结合转录因子AtbZIP60及其靶标BiP3启动子的分析。
