Molecular tools for studying the Arabidopsis transcription factor ORFeome

基因组学方法已经产生了大量的拟南芥开放阅读框（ORF）集合。然而，需要工具来在功能上对这个ORFeome进行编译。在此，我们基于现有的1200个全长cDNA的转录因子（TF）ORFeome提出了两个技术平台的开发：（1）我们开发了一种新的批量程序，以同时将GATEWAY®标记的cDNA重组到植物表达载体中。该库已被应用于建立拟南芥TF ORF过表达（AtTORF-Ex）种子库，该种子库包含接近完整的115个AP2/ERFs TF的集合。作为这个项目的一部分，我们试图扩大这个库高达1000转基因株系覆盖约三分之二的拟南芥TF ORFeome。该资源将用于筛选介导增强胁迫耐受性的TF。(2)建立了一个基于高通量微量滴定板的原生质体转染系统，该系统将用于分析TF ORFeome（原生质体反式激活系统：PTA）的反式激活特性。通过提供质粒库，可以快速鉴定那些调节特定启动子的TF。这些分子工具将显着提高AFGN财团和拟南芥社区进行的TF的功能研究。

Genomic approaches have generated large Arabidopsis open reading frame (ORF) collections. However, tools are required to functionally characterise this ORFeome. In here we propose the development of two technology platforms based on an existing transcription factor (TF) ORFeome of 1200 full-length cDNAs: (1) We have developed a novel batch procedure to simultaneously recombine GATEWAY®-tagged cDNAs into a plant expression vector. This pool has been applied to build up an Arabidopsis thaliana TF ORF over expressing (AtTORF-Ex) seed stock library harbouring a near-complete collection of 115 AP2/ERFs TF. As part of this project we attempt to enlarge this library up to 1000 transgenic lines covering around two thirds of the Arabidopsis TF ORFeome. This resource will be used to screen for TFs mediating enhanced stress tolerance. (2) A high-throughput microtiter plate based protoplast transfection system has been set up which will be used to analyse the trans activation properties of the TF ORFeome (Protoplast Trans Activation System: PTA). By providing plasmid pools, those TFs can be rapidly identified, which are regulating a particular promoter. These molecular tools will significantly improve functional studies on TFs performed by the AFGN consortium and the Arabidopsis community.