Xu, Wenjia
- Institut national de la recherche agronomique (INRA)
Sammanfattning
Large-scale analysis of transcription factorcis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression. Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway (R) vectors were specifically designed, although standard binary vectors can also be employed. As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified. This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter.
Nyckelord
cis-element; cytometry; flavonoids; Physcomitrella patens; protoplasts; transcription factor
Publicerad i
New Phytologist
2012, volym: 193, nummer: 2, sidor: 504-512
SLU författare
UKÄ forskningsämne
Genetik och genomik
Publikationens identifierare
- DOI: https://doi.org/10.1111/j.1469-8137.2011.03936.x
Permanent länk till denna sida (URI)
https://res.slu.se/id/publ/84782