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.

奖项摘要：这个试点项目将开发一种高通量策略来分析功能未知的拟南芥基因产物的本地表达模式和亚细胞定位。这一名为全长蛋白荧光标签(FTFLP)的策略将包括五个主要步骤：(1)选择“功能未分配”的拟南芥基因，预测其蛋白质结构和合适的荧光标签插入位置(2)分两部分扩增每个基因，两部分之间的连接对应于我们选择的荧光标签插入位置(3)引入荧光标签，利用三重重叠PCR的方法将黄色荧光蛋白(YFP)插入到双元载体中(4)将PCR产物插入到双元载体中(5)建立转基因拟南芥品系，并对每种标记蛋白的表达模式和细胞内定位进行分析。作为一种试点方法，该项目旨在分析具有统计意义的大量基因，以支持随后更广泛的研究的适用性。为此，从总共约8,000个未知基因中选择了大约800个基因(列在已经开始运作的项目网站http://arabidopsis.org/info/2010_projects/proteintagging.html)上)。根据下列顺序应用的标准选择了这个试点列表：1)具有匹配的全长cDNA，2)被标注为“未知蛋白质”或“假定蛋白质”，以及3)没有任何基因本体论标注。所选择的基因反映了所有未知拟南芥基因在植物专一性、预测结构域和/或基因家族信息以及匹配的全长cDNA序列的可用性方面的多样性。FTFLP作为功能蛋白质组学工具有三个显著的优点：它关注未知功能的基因，它产生的全长蛋白是内部标记的，更有可能表现出准确的细胞内定位，它利用天然启动子来确定组织特异性。将向研究界提供三项成果：1)在其固有启动子下含有每个基因全长序列并带有YFP标签的表达载体，2)表达每个结构的拟南芥转基因系，以及3)一个网站和一个可搜索的数据库，其中包含关于这些系和结构的信息，包括突出显示的基因序列和标签位置，载体结构信息，蛋白质表达模式和细胞内定位的图像和文本描述，以及实验、分析和解释中的规程和标准操作程序。此外，参考蛋白质亚细胞定位图将使用已知的细胞内靶向的荧光标记蛋白质来构建。这些资源将通过两个不受限制的场所向公众开放：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，而基因序列和表达以及包括荧光显微镜图像在内的亚细胞定位数据将通过整合到拟南芥信息资源(TAIR)的项目网站传播。重要的是，随着可交付成果的出现，通过ABRC和TAIR分别分享该项目的资源和成果的工作将持续进行。将通过Bionet Usenet新闻组和平行电子邮件列表等电子媒体宣布新资源的可获得性。该项目通过大规模描述未知拟南芥基因的表达和亚细胞定位，大大推进了2010年项目的总体目标。如果没有这样的知识，我们对拟南芥生物学的理解将是明显不完整的。此外，该项目还对社会和科学产生了更广泛的影响。一旦这一试点项目证明了拟议方法的可行性，它将成为开发实验室课程的基础，用于高中学生和教师的细胞生物学培训，以及CSHL DNA学习中心的初级研究人员和每年一次的拟南芥分子遗传学课程，以及一年两次的UCR植物细胞生物学课程。最后，与社区大学合作的教学外展项目将让本科生参与暑期研究。因此，我们的计划将在实验室和课堂上将基因组方法与细胞生物学联系起来，并产生重要的新信息和工具来表征拟南芥蛋白质组。