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Gene Expression and Transcription Factor Binding Tests Using Mutated-Promoter Reporter Lines

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Plant Transcription Factors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1830))

Abstract

To control the expression of their target genes, plant transcription factors bind to specific DNA sequences (called cis-elements) adjacent to the genes they regulate, thereby promoting or blocking the recruitment of RNA polymerase. Functional analysis of cis-elements is therefore essential for understanding transcriptional regulation, which underlies developmental programs and environmental responses. Using transgenic promoters containing mutations in their cis-elements, the roles of cis-elements in both transcriptional activity and transcription factor binding can be analyzed. To generate mutated promoters, site-directed mutagenesis is used. Site-directed mutagenesis is an in vitro method that confers the desired mutation in a target through performing PCR of native DNA using a mutated oligonucleotide primer. In this chapter, we describe detailed protocols for cloning of promoter regions, PCR-based site-directed mutagenesis, the generation of Arabidopsis transgenic lines, and expression analysis. In addition, we describe an in vivo method to test the binding of transcription factors to promoters with wild-type or mutated cis-elements. This protocol mainly focuses on the use of transgenic lines generated by site-directed mutagenesis, but it can readily be adapted for use with lines generated by CRISPR/Cas9.

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Acknowledgments

This work was supported by grants from the Japan Science and Technology Agency ‘Precursory Research for Embryonic Science and Technology’ to N.Y. and A.N-Y; grants to N.Y. from the JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (no. 16H01468), the NAIST foundation, the Sumitomo Foundation and the Takeda Science Foundation; the Cross-ministerial Strategic Innovation Promotion Program to A.N-Y.

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Authors and Affiliations

  1. Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

    Ayako Nishizawa-Yokoi

  2. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan

    Ayako Nishizawa-Yokoi & Nobutoshi Yamaguchi

  3. Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan

    Nobutoshi Yamaguchi

Authors
  1. Ayako Nishizawa-Yokoi
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  2. Nobutoshi Yamaguchi
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Correspondence to Nobutoshi Yamaguchi .

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Editors and Affiliations

  1. Plant Stem Cell Regulation and Floral Patterning Laboratory, Nara Institute of Science and Technology, Ikoma, Nara, Japan

    Nobutoshi Yamaguchi

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Nishizawa-Yokoi, A., Yamaguchi, N. (2018). Gene Expression and Transcription Factor Binding Tests Using Mutated-Promoter Reporter Lines. In: Yamaguchi, N. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 1830. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8657-6_17

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  • DOI: https://doi.org/10.1007/978-1-4939-8657-6_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8656-9

  • Online ISBN: 978-1-4939-8657-6

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