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

摘要：该试点项目将开发一种高通量策略来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。这种全长蛋白荧光标记（FTFLP）策略将包括五个主要步骤：(1)选择拟南芥“功能未分配”基因，预测其蛋白结构和合适的荧光标签插入位点(2)将每个基因分两部分扩增，两部分的连接点对应我们选择的荧光标签插入位点(3)引入荧光标签；(4)将PCR产物插入到二元载体中(5)转基因拟南芥系的制备，并分析各标记蛋白的表达模式和细胞内定位。作为一种试点方法，该项目旨在分析具有统计意义的基因数量，以支持后续更广泛研究的适用性。为此，从总共约8000个未知基因中选择了大约800个基因（已在运行的项目网站http://arabidopsis.org/info/2010_projects/proteintagging.html上列出）。该试点列表是根据以下顺序应用标准选择的：1)具有匹配的全长cDNA， 2)注释为“未知蛋白质”或“推定蛋白质”，3)没有任何基因本体注释。所选基因反映了所有未知拟南芥基因在植物特异性、预测结构域和/或基因家族信息以及匹配全长cDNA序列的可获得性方面的多样性。FTFLP作为功能蛋白质组学的工具有三个显著的优势：它专注于功能未知的基因，它产生内部标记的全长蛋白，更有可能表现出忠实的细胞内定位，它利用天然启动子使我们能够确定组织特异性。将向研究界提供三项成果：1)表达载体包含每个基因在其天然启动子下的全长序列，并以YFP标记，两侧有独特的限制性位点；2)表达每个构建体的拟南芥转基因系；3)一个网站和一个可搜索的数据库，包含关于这些系和构建体的信息，包括引物和标记位点位置突出的基因序列、载体构建体信息；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，分析和解释的协议和标准操作程序。此外，参考蛋白亚细胞定位图将使用已知的细胞内靶向荧光标记蛋白构建。这些资源将通过两个不受限制的渠道向公众提供：DNA构建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、表达和亚细胞定位数据，包括荧光显微镜图像，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，分别通过ABRC和TAIR共享项目的资源和成果将持续进行。将通过诸如Bionet USENET新闻组和平行电子邮件列表等电子媒体宣布是否提供新资源。该项目通过大规模表征未知拟南芥基因的表达和亚细胞定位，显著推进了2010年项目的总体目标。如果没有这些知识，我们对拟南芥生物学的理解将明显不完整。此外，该项目对社会和科学具有更广泛的影响。一旦这个试点项目证明了所提出方法的可行性，它将成为开发一个实验室课程的基础，用于高中学生和教师的细胞生物学培训，以及CSHL DNA学习中心和每年一次的拟南芥分子遗传学课程的初级研究人员，以及两年一次的UCR植物细胞生物学课程。最后，一项与社区学院合作的教学拓展计划将让本科生参与暑期研究。因此，我们的项目将在实验室和课堂上连接基因组方法与细胞生物学，并产生重要的新信息和工具来表征拟南芥蛋白质组。

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.