Arabidopsis 2010: The Arabidopsis Gene Collection/ORFeome Project

拟开展的研究项目涉及植物生物学研究工具和资源的开发。利用各种计算方法对125 mb拟南芥基因组的DNA序列进行了阐明，揭示了大约25,000个基因的存在。为了使这些大量的信息有用，需要对每个转录单位有更精确的了解。为了满足这一需求，我们启动了一个ORFeome项目，以获得拟南芥中每个编码开放阅读框（ORF）的蛋白质的克隆。该项目的目标是通过实验验证该标注，并构建10500个拟南芥基因的全长ORF克隆。本研究对30%的标注基因进行了实验验证，发现目前的基因组标注正确率只有68%。此外，该项目还制作了一套10,500个公开可用的ORF克隆，用于植物功能和蛋白质组学研究。这个为期两年的2010年项目的近期目标是通过全长cDNA测序实验验证另外3330个基因的注释，并为新的转录单位构建/测序另外3333个ORF克隆。我们将重点关注已知被转录的基因，即所谓的“注释表达基因”，但无法获得全长ORF克隆。利用RT-PCR技术构建orf，并将其亚克隆到通用重组载体体系中。转录单元序列信息将存入GenBank，并显示在我们的网络数据库（http;//signal.salk.edu）上。该项目生产的无性系将被存放在俄亥俄州立大学拟南芥生物资源中心。我们将与美国、欧洲和日本的研究人员合作，最大限度地提高新基因向公众开放的速度。”该项目开发的资源将被大量研究人员广泛使用，并将为依赖全基因组信息的各种项目提供基础。该提案的一个重要特点是，一旦生产出所有的ORF克隆和序列，它们将免费提供给研究界。该计划的受益者将是整个植物生物学界，因为序列信息和克隆将为植物生物学家提供阐明拟南芥蛋白质组功能所需的基本试剂。该项目的另一个重要特点是对本科生进行生物信息学和基因组学方法的培训。该研究的完成将为植物生物学界提供重要的资源，使未来的各种研究工作成为可能，如全基因组蛋白质阵列和快速创建异位表达使用任何启动子调控的任何cDNA的植物。这些使能工具和技术对农业的长期影响预计将是深远的，为构建具有优越农艺性状的新植物物种提供基础知识。

Summary of the Proposed Project The proposed study pertains to the development of research tools and resources for the plant biology community. The elucidation of the DNA sequence of the 125-Mb Arabidopsis genome revealed the presence of approximately 25,000 genes using various computational approaches. In order for this vast amount of information to be useful, a more precise understanding of each of the transcriptional units is required. To fulfill this need, we initiated an ORFeome project to obtain clones for each protein coding open-reading-frame (ORF) in Arabidopsis. The project goals were to experimentally verify the annotation and construct full-length ORF clones for 10,500 of the Arabidopsis genes. This study resulted in the experimental verification of 30% of the annotated genes and revealed that the current genome annotation is only 68% correct. In addition, the project produced a set of 10,500 publically available ORF clones for plant functional and proteomics studies. The immediate near tem goal of this two-year 2010 project is to experimentally verify the annotation of an additional 3,330 genes by full-length cDNA sequencing and the construction/sequencing of 3,333 additional ORF clones for new transcriptional units. We will focus on genes that are known to be transcribed, so called "annotated expressed genes" but for which full-length ORF clones are not available. ORFs will be constructed using RT-PCR and they will be subcloned into universal recombination vector system. Transcription unit sequence information will be deposited in GenBank and displayed on our web accessible database (http;//signal.salk.edu). Clones produced by the project will be deposited with the Arabidopsis Biological Resource Center at Ohio State University. We will coordinate with researchers in the US, Europe and Japan to maximize the rate at which new genes become available to the public.Broader Impacts The resources developed by this project will be widely available to a large number of researchers and will provide the basis for a wide variety of projects that rely upon whole genome information. An important feature of this proposal is that all of the ORF clones and sequences will be made freely available to the research community as soon as they are produced. The beneficiaries of this program will be the entire plant biology community as both the sequence information and the clones will provide plant biologists with essential reagents necessary to elucidate the functions of the Arabidopsis proteome. Another important feature of the project is the training of undergraduate students in bioinformatics and genomic methodologies. The completion of the proposed research will provide an important resource to the plant biology community, enabling a variety of future research endeavors such as whole genome protein arrays and the rapid creation of plants that ectopically express any cDNA that is regulated using any promoter of choice. 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 species with superior agronomic traits.