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

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.