RESUMO
Protein-DNA interactions are determinant of the regulation of gene expression in living organisms. Luminescence studies have been used in a wide range of techniques to identify how gene transcription can be regulated by proteins such as transcription factors (TFs). Despite the great advances in the use of luciferases as reporters in the performance of this mechanism, some of them still have disadvantages that have been tried to be solved by the generation of new luciferases that induce a more stable and perfectly visualizable reaction. NanoLuc is a recently described luciferase that has been characterized by its efficient, stable, and powerful luminescence. These qualities have been considered to create a new and efficient reporter system to detect protein-DNA interactions. In this chapter, we take advantage of NanoLuc and describe its use in a reliable procedure to detect protein-DNA interactions in Nicotiana benthamiana extracts and entire leaves.
Assuntos
Nicotiana , Fatores de Transcrição , Ativação Transcricional , Luciferases/genética , Luciferases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
The phytohormone cytokinin is crucial for plant growth and development. The site of action of cytokinin in the plant is dependent on the expression of the cytokinin receptors. In Arabidopsis, there are three cytokinin receptors that present some overlap in expression pattern. Functional studies demonstrated that the receptors play highly redundant roles but also have specialized functions. Here, we focus on gynoecium development, which is the female reproductive part of the plant. Cytokinin signaling has been demonstrated to be important for reproductive development, positively affecting seed yield and fruit production. Most of these developmental processes are regulated by cytokinin during early gynoecium development. While some information is available, there is a gap in knowledge on cytokinin function and especially on the cytokinin receptors during early gynoecium development. Therefore, we studied the expression patterns and the role of the cytokinin receptors during gynoecium development. We found that the three receptors are expressed in the gynoecium and that they have redundant and specialized functions.