The Arabidopsis Transcription Factor ORFeome and downstream genomic application

拟南芥转录因子ORFeome及其下游基因组应用

• 批准号: 7853305
• 负责人: Joseph R Ecker
• 金额: $ 108.92万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7853305
• 关键词: Alzheimer' s Disease; Arabidopsis; Binding; Biochemical; Biological Assay; Biological Process; Biomass; Biomedical Research; Cloning; Code; Collaborations; Collection; Communities; DNA; DNA Resequencing; DNA-Protein Interaction; Deposition; Detection; Development; Disease; Eligibility Determination; Epitopes; Equipment; Food; Galactosidase; Gene Expression; Genes; Genomic Library; Genomics; Glycerol; Gold; Health; Human; Hybrids; Institutes; Knowledge; Laboratories; Laboratory Research; Libraries; Luciferases; Maintenance; Malignant Neoplasms; Malnutrition; Measurement; Measures; Molecular; Molecular Biology; Molecular Target; Nucleic Acid Regulatory Sequences; Organism; Parkinson Disease; Pathway interactions; Plants; Procedures; Production; Productivity; Proteins; Protocols documentation; Reading; Recombinant Proteins; Regulation; Reporter; Reporting; Research; Resources; Role; Structure; Systems Biology; Technology; Testing; Transcription Initiation Site; Transgenic Organisms; Ubiquitin; Vertebral column; Visualization software; Yeasts; bacterial vector; base; chromatin immunoprecipitation; cost; functional genomics; innovation; novel; overexpression; promoter; protein expression; protein protein interaction; research study; tool; transcription factor; vector; web site; yeast two hybrid system

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.

描述（由申请人提供）：转录因子（TF）在控制基因表达方面发挥着至关重要的作用，探索其分子靶标、结合伴侣和调控模式对于理解任何植物生物过程至关重要。拟南芥为开发用于研究转录因子功能的大规模基因组方法提供了一些独特的优势，例如生成大型转基因集合的容易性和低成本，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的倾向。我们实验室最近的一份报告充分说明了此类策略的潜力，其中通过使用完整 TF 集合的一小部分，识别出了一种新颖的时钟组件。出于这些原因，我们开始从不同的 ORFeome 资源（Salk、Pekin-Yale、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，我们对不同克隆进行重新测序的结果显示，这些集合之间存在很大的重叠，并且有数百个错误标记或缺失的克隆，导致最终覆盖率接近所有拟南芥转录因子和调节子的 75%。在这里，我们建议生成一个包含每个拟南芥 TF 的同质黄金标准 GATEWAYTM 兼容集合。如果可能，将使用可用的 ORFeomes 作为模板资源，将相应的编码序列克隆到同一载体中。其余 25% 的缺失片段将通过不同的互补扩增方案生成，并随后克隆到同一载体主链中。此外，我们建议创建并向社区分发九个可供应用的基因组集合，其中每个 TF 与不同标签融合，用于多种应用。这九个集合将允许 (1) 野生型植物以及 EAR 或 VP64 翻译融合的过表达筛选，(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，以及 (5) 细菌重组蛋白表达。此外，我们与索尔克研究所的 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。将生成标有所选表位的 TF 集合。最后，我们建议设计一个简单的方案，以合理的成本对完整的 TF 集合进行酵母单杂交筛选。我们坚信这些资源有潜力极大地加强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最终将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。