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: 7939663
• 负责人: Joseph R Ecker
• 金额: $ 91.09万
• 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 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7939663
• 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; abstracting; 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具有关键作用，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学过程至关重要。拟南芥提供了一些独特的优势，为发展大规模的基因组方法，如容易和低成本产生大的转基因集合，或大多数基因调控区被限制到一个短的区域上游的转录起始位点的功能研究。我们实验室最近的一份报告充分体现了这些类型策略的潜力，其中通过使用完整TF集合的一部分，识别出了一种新型时钟组件。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因子的75%。在这里，我们建议产生一个同质的金标准GATEWAYTM兼容的收集包含每个拟南芥TF。在可能的情况下，使用可用的ORFeomes作为模板资源，将相应的编码序列克隆到相同的载体中。剩余的25%缺失的片段将通过以下不同的互补扩增方案产生，随后克隆到相同的载体骨架中。此外，我们建议创建和分发给社会，九个应用程序准备基因组集合包含每个TF融合不同的标签，用于多种应用程序。这九个集合将允许（1）在野生型植物中的过表达筛选以及VP 64或VP 64翻译融合，（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）亚细胞定位和（5）细菌重组蛋白表达。此外，与索尔克研究所的Joe Ecker博士合作，我们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。将产生用所选表位标记的TF的集合。最后，我们建议设计一个简单的协议，以合理的成本进行酵母单杂交筛选与完整的TF集合。我们坚信这些资源有潜力大大提高拟南芥和其他作物物种的研究。随着知识缺口的填补，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。