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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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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