The Arabidopsis Transcription Factor ORFeome and downstream genomic application

描述（申请人提供）：转录因子（Transcription factors， TF）在控制基因表达中起着至关重要的作用，探索其分子靶点、结合伙伴和调控模式对理解任何植物生物学过程都是至关重要的。拟南芥为研究tf功能的大规模基因组方法的发展提供了一些独特的优势，例如容易和低成本地产生大量转基因集合，或者大多数基因调控区域被限制在转录起始位点上游的短区域。这些类型策略的潜力在我们实验室最近的一份报告中得到了很好的例证，通过使用完整TF收集的一小部分，确定了一种新的时钟组件。由于这些原因，我们开始从不同的ORFeome资源（Salk，北京-耶鲁，REGIA， TIGR和RIKEN）收集所有可用的拟南芥TF，以生成完整的TF集合。然而，我们对不同克隆的重测序结果显示，收集的样本和数百个错误标记或缺失的样本之间存在很大的重叠，导致最终覆盖了近75%的拟南芥转录因子和调控因子。在这里，我们建议生成一个包含所有拟南芥TF的同质金标准GATEWAYTM兼容集合。在可能的情况下，使用可用的orfeome作为模板资源，将相应的编码序列克隆到相同的向量中。其余25%的缺失基因将通过不同的互补扩增方案产生，随后克隆到同一载体主干上。此外，我们建议创建并分发给社区，九个应用就绪的基因组集合包含每个TF融合不同的标签，用于多种应用。这9个集合将允许(1)在野生型植物以及EAR或VP64翻译融合物中进行过表达筛选，(2)蛋白质-蛋白质相互作用筛选，(3)蛋白质- dna相互作用筛选，(4)亚细胞定位，(5)细菌重组蛋白表达。此外，我们与Salk研究所的Joe Ecker博士合作，建议测试和比较适合进行ChIP-seq实验的不同蛋白质表位标签的效率。将生成带有所选表位标记的TF集合。最后，我们建议设计一种简单的方案，以合理的成本进行酵母单杂交筛选，并收集完整的TF。我们坚信这些资源有潜力极大地加强对拟南芥和其他作物物种的研究。随着知识空白的填补，对拟南芥转录网络的研究将最终帮助我们了解多细胞生物的生化复杂性，并对生物医学研究界产生积极影响。

DESCRIPTION (provided by applicant): Transcription factors (TF) have a crucial role in controlling gene expression, and exploring their molecular targets, binding partners and mode of regulation is essential to understand any plant biological process. Arabidopsis offers some unique advantages for the development of large-scale genomic approaches for the study of TFs function such as the ease and low cost to generate large transgenic collections, or the propensity of most gene-regulatory regions to be circumscribed to a short region upstream the transcription start site. The potential of these types of strategies is greatly exemplified in a recent report from our laboratory where, by using a fraction of the full TF collection, a novel clock component was identified. For these reasons, we started gathering all available Arabidopsis TFs from the different ORFeome resources (Salk, Pekin-Yale, REGIA, TIGR and RIKEN) to generate a complete TF collection. However, our findings from resequencing the different clones revealed a large overlap between the collections and several hundred mislabeled or missing ones, resulting in a final coverage close to 75% of all the Arabidopsis transcription factors and regulators. Here, we propose to generate an homogenous gold standard GATEWAYTM compatible collection containing every Arabidopsis TF. The corresponding coding sequences will be cloned in the same vector using the available ORFeomes as the template resource when possible. The remaining 25% missing ones will be generated by following different complementary amplification protocols and subsequently cloned in the same vector backbone. In addition, we propose to create and distribute to the community, nine application-ready genomic collections containing each TF in fusion with different tags for a multitude of applications. These nine collections will allow (1) overexpression screens in plants of wild type as well as EAR or VP64 translational fusions, (2) protein-protein interaction screens, (3) protein-DNA interaction screens, (4) subcellular localization and, (5) bacterial recombinant protein expression. In addition, in collaboration with Dr. Joe Ecker at the Salk Institute, we propose to test and compare the efficiency of different protein epitope-tags suitable to perform ChIP-seq experiment. A collection of TF tagged with the selected epitope will be generated. Finally, we propose to devise a simple protocol to perform yeast one-hybrid screens with full TF collections at a reasonable cost. We strongly believe these resources have the potential to greatly enhance research in Arabidopsis and other crop species. As the knowledge gap is being filled, the study of transcriptional networks in Arabidopsis will ultimately help us understand the biochemical complexity of multicellular organisms and positively impact the biomedical research community.