Arabidopsis 2010: Large-Scale Fluorescent Tagging of Full-Length Genes to Characterize Native Expression Patterns and Subcellular Targeting of Arabidopsis Proteins of Unknown Funct

拟南芥 2010：全长基因的大规模荧光标记，以表征未知功能拟南芥蛋白的天然表达模式和亚细胞靶向

• 批准号: 0210992
• 负责人: Vitaly Citovsky
• 金额: $ 158万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0210992&HistoricalAwards=false
• 关键词: Arabidopsis; 2010; Large; Scale; Fluorescent

AWARD ABSTRACT:This pilot project will develop a high-throughput strategy to analyze native expression patterns and subcellular localization of Arabidopsis gene products of unknown function. This strategy, Fluorescent Tagging of Full-Length Proteins (FTFLP), will comprise five major steps: (1) Selection of "functionally unassigned" Arabidopsis genes and prediction of their protein structure and suitable site for fluorescent tag insertion (2) Amplification of each gene in two parts, with the junction between the two parts corresponding to our chosen insertion site for the fluorescent tag(3) Introduction of the fluorescent tag, yellow fluorescent protein (YFP) using a triple overlap PCR approach (4) Insertion of PCR products into binary vectors (5) Production of transgenic Arabidopsis lines and analysis of expression pattern and intracellular localization for each tagged protein. As a pilot approach, the project aims to analyze a statistically significant number of genes to support the applicability to a subsequent wider study. To this end, approximately 800 genes (listed at the already operational project website http://arabidopsis.org/info/2010_projects/proteintagging.html) were selected from a total of ca. 8,000 unknown genes. This pilot list was chosen based on the following sequentially-applied criteria: 1) have matching full-length cDNA, 2) are annotated as 'unknown protein' or 'putative protein', and 3) do not have any Gene Ontology annotations. The selected genes reflect the diversity of all the unknown Arabidopsis genes with respect to plant specificity, predicted domain and/or gene family information, and availability of matching full-length cDNA sequences.FTFLP as a tool for functional proteomics offers three significant advantages: it focuses on genes of unknown function, it produces internally-tagged full length proteins that are more likely to exhibit faithful intracellular localization, and it utilizes native promoters to allow us to determine tissue specificity. Three deliverables will be offered to the research community: 1) Expression vectors harboring full-length sequences for each gene under its native promoter and tagged with YFP flanked by unique restriction sites,2) Arabidopsis transgenic lines expressing each construct, and 3) A website and a searchable database containing information about the lines and constructs, including the gene sequences highlighted with positions of primers and tagging sites, vector construct information, images and text descriptions of the protein expression pattern and intracellular localization, and protocols and standard operation procedures in experimentation, analysis, and interpretation. Also, a Reference Protein Subcellular Localization Map will be constructed using fluorescently-tagged proteins with known intracellular targeting. These resources will be available to the public through two unrestricted venues: DNA constructs and transgenic seeds will be distributed through the Arabidopsis Biological Resource Center (ABRC) whereas gene sequences and expression and subcellular localization data, including fluorescence microscopy images, will be disseminated via the project website integrated into The Arabidopsis Information Resource (TAIR). Importantly, this sharing of the resources and results of this project through ABRC and TAIR, respectively, will take place on a continuous basis as the deliverables become available. Announcements on the availability of new resources will be made through such electronic media as the Bionet USENET newsgroups and parallel e-mail lists.This project significantly advances the overall objectives of the 2010 Project by characterizing on a large scale the expression and subcellular localization of unknown Arabidopsis genes. Our understanding of Arabidopsis biology will be glaringly incomplete without such knowledge. In addition, this project has a broader impact on the society and science. Once this pilot project demonstrates the feasibility of the proposed approach, it will serve a basis for developing a laboratory curriculum for use in cell biology training of high school students and teachers as well as beginning investigators at the CSHL DNA Learning Center and the annual Arabidopsis Molecular Genetics Course, and at the biannual UCR Plant Cell Biology course. Finally, a teaching outreach program with community colleges will involve undergraduates in summer research. Thus, our program will bridge genomic approaches with cell biology in the laboratory and classroom, and generate important novel information and tools to characterize the Arabidopsis proteome.

奖励摘要：该试点项目将制定高通量策略，以分析未知功能的拟南芥基因产物的天然表达模式和亚细胞定位。 This strategy, Fluorescent Tagging of Full-Length Proteins (FTFLP), will comprise five major steps: (1) Selection of "functionally unassigned" Arabidopsis genes and prediction of their protein structure and suitable site for fluorescent tag insertion (2) Amplification of each gene in two parts, with the junction between the two parts corresponding to our chosen insertion site for the fluorescent tag(3) Introduction of the fluorescent tag, yellow荧光蛋白（YFP）使用三重重叠PCR方法（4）将PCR产物插入二进制载体（5）生产转基因拟南芥线条以及每种标记蛋白的表达模式和细胞内定位的分析。作为试点方法，该项目旨在分析统计上重要的基因，以支持对随后的更广泛研究的适用性。为此，大约有800个基因（在已经运营的项目网站http://arabidopsis.org/info/2010_projects/proteintagging.html中列出）。 8,000个未知基因。该试点列表是根据以下顺序应用的标准选择的：1）具有匹配的全长cDNA，2）注释为“未知蛋白”或“推定蛋白”，而3）没有任何基因本体论注释。 The selected genes reflect the diversity of all the unknown Arabidopsis genes with respect to plant specificity, predicted domain and/or gene family information, and availability of matching full-length cDNA sequences.FTFLP as a tool for functional proteomics offers three significant advantages: it focuses on genes of unknown function, it produces internally-tagged full length proteins that are more likely to exhibit faithful intracellular localization, and it utilizes native启动子使我们能够确定组织特异性。 Three deliverables will be offered to the research community: 1) Expression vectors harboring full-length sequences for each gene under its native promoter and tagged with YFP flanked by unique restriction sites,2) Arabidopsis transgenic lines expressing each construct, and 3) A website and a searchable database containing information about the lines and constructs, including the gene sequences highlighted with positions of primers and tagging sites, vector construct information, images and text蛋白质表达模式和细胞内定位的描述，以及实验，分析和解释中的方案和标准操作程序。此外，将使用具有已知细胞内靶向的荧光标记蛋白来构建参考蛋白亚细胞定位图。这些资源将通过两个不受限制的场所向公众提供：DNA构建体和转基因种子将通过拟南芥生物资源中心（ABRC）分发，而基因序列和表达和表达和亚细胞定位数据（包括荧光显微镜图像）将通过项目网站进行散布，集成到拟南芥信息资源（Tair）中。重要的是，随着可交付成果的可用，分别通过ABRC和TAIR分别分别进行了该项目的资源和结果共享。关于新资源可用性的公告将通过诸如Bionet Usenet新闻组和并行电子邮件列表等电子媒体进行。该项目通过大规模表征不知名的拟南芥基因的表达和亚细胞定位来显着提高2010年项目的整体目标。没有这种知识，我们对拟南芥生物学的理解将是明显的不完整。此外，该项目对社会和科学有更广泛的影响。一旦该试点项目证明了拟议方法的可行性，它将为开发实验室课程提供基础，用于在高中生和老师的细胞生物学培训中使用，并在CSHL DNA学习中心和年度拟南芥分子遗传学课程以及Bianal Mianal UCR植物细胞生物学课程中开始研究人员。最后，与社区大学的教学外展计划将涉及夏季研究。因此，我们的程序将在实验室和教室中使用细胞生物学桥接基因组方法，并生成重要的新颖信息和工具来表征拟南芥蛋白质组。