Chapter title |
Gene Expression and Transcription Factor Binding Tests Using Mutated-Promoter Reporter Lines
|
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Chapter number | 17 |
Book title |
Plant Transcription Factors
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-8657-6_17 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8656-9, 978-1-4939-8657-6
|
Authors |
Ayako Nishizawa-Yokoi, Nobutoshi Yamaguchi, Nishizawa-Yokoi, Ayako, Yamaguchi, Nobutoshi |
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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