An efficient protocol for functional studies of apple transcription factors using a glucocorticoid receptor fusion system
2020
Estevan, Joan | Gomez-Jimenez, Sara | da Silveira Falavigna, Vítor | Camuel, Alicia | Planel, Lisa-Fatimatou | Costes, Evelyne | Andres, Fernando
Premise: We report a protocol for studying the function of apple (Malus ×domestica) transcription factors based on the glucocorticoid receptor (GR) system, which allows the dexamethasone (DEX)-mediated activation of plant transcription factors to monitor the expression levels of their potential target genes. Methods and Results: Apple leaves are transformed with a vector that allows the expression of the studied transcription factor (i.e., FLOWERING LOCUS C [MdFLC]) fused to GR. Calli derived from the transformed leaves are treated with DEX and cycloheximide, a protein synthesis inhibitor. Compared with other methods, combining the GR system with cycloheximide treatments enables the differentiation between direct and indirect transcription factor target genes. Finally, the expression levels of putative MdFLC target genes are quantified using quantitative reverse transcription PCR. Conclusions: We demonstrate the efficiency of our GR system to study the function of apple transcription factors. This method is accessible to any laboratory familiar with basic molecular cloning procedures and the apple leaf–mediated agro-transformation technique.
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