Tools and Technologies to Enable Genome-Wide Screens in Arabidopsis

在拟南芥中实现全基因组筛选的工具和技术

• 批准号: 1122250
• 负责人: Joseph Ecker
• 金额: $ 354.34万
• 依托单位: The Salk Institute for Biological Studies
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1122250&HistoricalAwards=false
• 关键词: Tools; Technologies; Enable; Genome; Wide

Intellectual Merit: This project aims to develop genome-wide experimental tools and technologies for analyzing gene regulation and function in Arabidopsis. Computational analysis of the initial genome sequence of Arabidopsis thaliana, completed in the year 2000, provided evidence for the existence of approximately 25,500 protein-coding genes. Intensive efforts during the past ten years to experimentally annotate this genome sequence have now identified 31,128 genes, which include additional protein-coding genes as well as non-coding RNA genes. The identification of T-DNA insertion mutations in all Arabidopsis genes has been an on-going aim of worldwide functional genomics programs. Analysis of the available set of TDNA mutants reveals that ~24,000 additional mutant alleles are needed to create a comprehensive homozygous mutant collection. This research specifically addresses the following goals: to identify two genetically stable loss-of-function mutations in all Arabidopsis genes and to complete the goal of isolating two homozygous alleles for every gene in the genome. llumina paired-end deep sequencing will be used to identify mutations in these "missing" genes, thereby allowing completion of the "unimutant" for the annotated set of Arabidopsis genes. Further development of the T-DNA-Seq method for large scale capture and sequencing of ~ one million T-DNA insertion sites will allow identification of the inventory of essential genes as well as provide insights into the mechanism of T-DNA integration and associated gene silencing events. Broader Impacts: The genomic resources developed by this project will be widely available to a large number of researchers and will provide the basis for a variety of research projects that rely upon whole genome information. Completion of the proposed research will provide a new important resource for the plant biology community, enabling a variety of genome-wide mutant screens for any visible phenotype of interest. An important feature of this research is that all of the mutant plants/populations will be available to the research community as soon as they are produced. The beneficiaries of this program will be the entire plant biology community, providing essential reagents necessary to elucidate the functions of the Arabidopsis genes. Additionally, the new technology developed to rapidly and inexpensively index insertion mutants by next generation sequencing in any plant or animal will have applications far beyond Arabidopsis research. The long-term impact of these enabling tools and technologies on agriculture is expected to be profound, providing fundamental knowledge for the construction of new plant varieties with superior agronomic traits. An equally important aspect of our research program is the hands-on training in plant genome research that will be provided at a variety of levels, including outreach to minority high school and undergraduate students.

智力优势：该项目旨在开发全基因组实验工具和技术，用于分析拟南芥中的基因调控和功能。2000年完成的对拟南芥初始基因组序列的计算分析为大约25，500个蛋白质编码基因的存在提供了证据。在过去的十年里，人们对这一基因组序列进行了大量的实验性注释，现已确定了31，128个基因，其中包括额外的蛋白质编码基因和非编码RNA基因。拟南芥基因中T-DNA插入突变的鉴定一直是世界范围内功能基因组学研究的目标。对可用的TDNA突变体集的分析揭示，需要约24，000个额外的突变等位基因来创建全面的纯合突变体集合。本研究具体涉及以下目标：在所有拟南芥基因中鉴定两个遗传稳定的功能丧失突变，并完成为基因组中的每个基因分离两个纯合等位基因的目标。llumina配对末端深度测序将用于鉴定这些“缺失”基因中的突变，从而允许完成拟南芥基因的注释集的“单突变体”。进一步开发T-DNA-Seq方法，用于大规模捕获和测序约一百万个T-DNA插入位点，将允许鉴定必需基因的库存，并提供对T-DNA整合机制和相关基因沉默事件的见解。更广泛的影响：该项目开发的基因组资源将广泛提供给大量研究人员，并将为依赖于全基因组信息的各种研究项目提供基础。拟议研究的完成将为植物生物学界提供一个新的重要资源，使各种全基因组突变体筛选任何可见的感兴趣的表型。这项研究的一个重要特点是，所有的突变植物/种群将提供给研究界，一旦他们产生。该计划的受益者将是整个植物生物学社区，为阐明拟南芥基因的功能提供必要的试剂。此外，通过下一代测序在任何植物或动物中快速和廉价地索引插入突变体的新技术将具有远远超出拟南芥研究的应用。这些使能工具和技术对农业的长期影响预计将是深远的，为构建具有上级农艺性状的新植物品种提供基础知识。我们研究计划的一个同样重要的方面是植物基因组研究的实践培训，将在各个层面提供，包括推广到少数民族高中和本科生。