Macromolecular Networks Underlying Chloroplast Biogenesis

主要研究者：爱丽丝·巴肯（俄勒冈州大学-尤金）CoPI：克拉斯·货车·维克（康奈尔大学）叶绿体是植物的一个重要特征，具有多种代谢功能，深刻影响植物产量和对环境胁迫的反应。光合作用是叶绿体存在的主要理由，涉及复杂的机制，其生物发生需要一系列功能和物理相关的基因表达和组装过程。该项目的目标是促进对DNA/RNA/蛋白质相互作用网络的理解，该网络是叶绿体中光合机构生物发生的基础。预计选择用于研究的大分子组装物富含未知功能的蛋白质，并将通过协同遗传学、蛋白质组学和核糖组学分析进行解剖。 玉米将是主要的实验生物，因为它的属性使它特别适合这些方法-它是一种重要的作物物种，也是了解C4光合作用的主要模型。与拟南芥的选择性比较将有助于将结果外推到单子叶植物和双子叶植物物种。叶绿体生物发生所需的核基因将通过高通量正向遗传策略发现，该策略将下一代测序技术与非光合玉米突变体的深入收集相结合。将突变体置于未充分研究的功能类别中的现有表型数据将用于选择100个品系用于该分析。 免疫纯化的大分子组装体的蛋白质组分将通过高灵敏度质谱法鉴定。抗体靶标将包括：与将多核糖体翻译成用于合成和装配叶绿体编码蛋白质的免疫纯化“工厂”相关的新生肽链;与叶绿体染色体相关的蛋白质;以及在叶绿体基因表达和蛋白质折叠/装配机构的界面处的蛋白质。新鉴定的蛋白质将通过反向遗传学和表型分析以及通过鉴定其核酸配体（如相关）进行功能表征。 最后，全基因组RNA/DNA免疫共沉淀分析将用于鉴定与通过遗传学和蛋白质组学方法鉴定的核酸相互作用蛋白相关的核酸，并探索细胞器专用RNA结合蛋白家族的功能。这项研究将与各级学生的教育相结合。 俄勒冈州大学将为高中生制定一项夏季研究计划，该计划以与本项目目标不可或缺的实验为中心，并为担任助教的本科生和研究生提供指导培训。 该项目将参与康奈尔大学/BTI现有的项目，为来自小型机构的本科生提供研究培训，并参加高中教师的课程开发研讨会。将产生的社区资源包括：（i）相互作用数据、蛋白质鉴定和遗传数据，所有这些都将提交给公共数据库储存库，并用于开发数百个玉米基因的功能注释;（ii）约100个具有鉴定的突变和表征的突变表型的突变玉米品系，以及数千个序列索引的转座子插入和相关的玉米原种;和（iii）玉米细胞器微阵列和各种叶绿体蛋白的抗体。相互作用数据将有助于社区努力开发高质量的植物相互作用组数据集。相互作用数据和基因注释将通过集中资源传播，包括Gramene（http：//www.gramene.org/）、MaizeGDB（http：//www.maizegdb.org/）、PlantGDB（http：//www.plantgdb.org/）和植物蛋白质组数据库（PPDB; http：//ppdb.tc.cornell.edu/）。玉米库存将提供给玉米遗传合作社库存中心进行分配。微阵列和抗体将根据要求按成本提供。

PI: Alice Barkan (University of Oregon - Eugene)CoPI: Klaas van Wijk (Cornell University)Chloroplasts are a defining feature of plants, with diverse metabolic functions that profoundly impact plant yield and response to environmental stress. Photosynthesis is the primary raison d'etre of the chloroplast, and involves complex machinery whose biogenesis requires a series of functionally and physically-connected gene expression and assembly processes. The goal of this project is to advance understanding of the network of DNA/RNA/protein interactions that underlie the biogenesis of the photosynthetic apparatus in chloroplasts. Macromolecular assemblies selected for study are anticipated to be rich in proteins of unknown function, and will be dissected through concerted genetic, proteomic, and ribonomic analyses. Maize will be the primary experimental organism because its attributes make it especially well suited for these methods - it is an important crop species and a prime model for understanding C4 photosynthesis. Selective comparisons with Arabidopsis will facilitate extrapolation of results to both monocot and dicot species. Nuclear genes required for chloroplast biogenesis will be discovered through a high-throughput forward-genetic strategy that combines next generation sequencing technologies with a deep collection of non-photosynthetic maize mutants. Existing phenotypic data that place mutants into understudied functional classes will be used to select 100 lines for this analysis. Protein components of immunopurified macromolecular assemblies will be identified through high sensitivity mass spectrometry. Antibody targets will include: nascent peptide chains associated with translating polysomes to immunopurified "factories" for the synthesis and assembly of chloroplast-encoded proteins; proteins associated with the chloroplast chromosome; and proteins at the interface of the chloroplast gene expression and protein folding/assembly machineries. Newly-identified proteins will be functionally characterized by reverse-genetics and phenotypic analyses, and by identification of their nucleic acid ligands (where relevant). Finally, genome-wide RNA/DNA co-immunoprecipitation assays will be used to identify the nucleic acids associated with nucleic-acid interacting proteins identified via the genetic and proteome approaches, and to explore the functions of organelle-dedicated RNA binding protein families.This research will be integrated with the education of students at all levels. A summer research program for high school students that centers on experiments integral to the goals of this project, and that provides mentorship training for undergraduate and graduate students who serve as teaching assistants will be developed at the University of Oregon. The project will participate in existing programs at Cornell/BTI to provide research training for undergraduate students from small institutions and to participate in curriculum development workshops for high school teachers. Community resources to be generated include: (i) interaction data, protein identifications, and genetic data, all of which will be submitted to public database repositories and used to develop functional annotations for several hundred maize genes; (ii) ~100 mutant maize lines with an identified mutation and characterized mutant phenotype, as well as several thousand sequence-indexed transposon-insertions and associated maize stocks; and, (iii) maize organellar microarrays and antibodies to various chloroplast proteins. The interaction data will contribute to community efforts to develop a high quality plant interactome data set. Interaction data and gene annotations will be disseminated through centralized resources that include Gramene (http://www.gramene.org/), MaizeGDB (http://www.maizegdb.org/), PlantGDB (http://www.plantgdb.org/), and the Plant Proteome Database (PPDB; http://ppdb.tc.cornell.edu/). Maize stocks will be provided to the Maize Genetics Coop Stock Center for distribution. Microarrays and antibodies will be made available at cost by request.