INRA AgroParisTech, IJPB, UMR 1318, INRA centre de Versailles, Versailles, France.
New Phytol. 2012 Jan;193(2):504-12. doi: 10.1111/j.1469-8137.2011.03936.x. Epub 2011 Oct 24.
• Large-scale analysis of transcription factor-cis-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® 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.
• 大规模分析植物转录因子顺式作用元件相互作用，或解析复杂的转录调控机制，都需要快速、稳健和可靠的基因表达定量系统。• 在这里，我们描述了一种用于转录因子瞬时表达分析的新系统，该系统利用快速简便的 Physcomitrella patens 原生质体生产和转染，结合流式细胞术对荧光蛋白（绿色荧光蛋白）进行定量。专门设计了两个小型和高拷贝的 Gateway®载体，尽管也可以使用标准的二元载体。• 作为概念验证，我们使用了拟南芥种子中参与原花青素生物合成的关键结构基因 BANYULS（BAN）的调控来进行研究。在 P. patens 中，BAN 的表达受由三个蛋白（TT2/AtMYB123、TT8/bHLH042 和 TTG1）组成的复合物激活，并像在拟南芥中一样被转录抑制因子 MYBL2 抑制。使用这种方法，我们鉴定了两个在原花青素积累细胞中特异性表达 BAN 所必需和充分的新调控序列。• 这种类似于杂交的植物系统成功地用于定量评估四个调节蛋白的转录活性，并鉴定它们在 BAN 启动子上的靶识别位点。
