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Gene Expression and Transcription Factor Binding Tests Using Mutated-Promoter Reporter Lines
Ayako Nishizawa-Yokoi1,2, Nobutoshi Yamaguchi3,4
1Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Methods in Molecular Biology (Clifton, N.J.)
|July 26, 2018
Summary
Understanding plant gene regulation requires analyzing cis-elements. This study details methods for mutating cis-elements in plant promoters to analyze transcription factor binding and gene expression, aiding in plant development and environmental response research.
Area of Science:
- Plant molecular biology
- Genetics
- Biochemistry
Background:
- Plant transcription factors regulate gene expression by binding to cis-elements near target genes.
- Functional analysis of cis-elements is crucial for understanding transcriptional regulation in plant development and environmental responses.
Purpose of the Study:
- To provide detailed protocols for analyzing cis-element function in plant gene regulation.
- To enable the study of transcription factor binding and transcriptional activity using mutated promoters.
Main Methods:
- Cloning of plant promoter regions.
- Polymerase chain reaction (PCR)-based site-directed mutagenesis to create promoter variants.
- Generation of Arabidopsis transgenic lines.
- Gene expression analysis.
- In vivo methods to assess transcription factor binding to wild-type and mutated cis-elements.
Main Results:
- Detailed protocols are provided for generating and analyzing transgenic plants with mutated cis-elements.
- The methods allow for the investigation of cis-element roles in both transcriptional activity and transcription factor binding.
- The described protocols can be adapted for use with CRISPR/Cas9-generated lines.
Conclusions:
- The described protocols offer a comprehensive approach to functionally analyze cis-elements in plant gene regulation.
- These methods are essential for dissecting the molecular mechanisms underlying plant development and responses to environmental stimuli.
- The study facilitates research into plant transcription factor-DNA interactions and their impact on gene expression.
Keywords:
ArabidopsisDNA-binding domainPlant transcription factorsSite-directed mutagenesiscis-ElementsMore Related Videos
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