Chromatin-based Control of Gene Expression in Maize and Arabidopsis

该奖项支持在染色质水平上控制基因表达的基因突变的产生。该项目的总体目标是识别和功能分析玉米(玉米)和拟南芥中有助于染色质水平控制基因表达的数百个基因中的大多数，如果不是全部的话。玉米是美国最重要的农作物，也是解决单子叶作物基本问题的主要模式系统，尤其是谷物。染色质是与DNA一起构成染色体的蛋白质物质。基因在染色体中表达的一个关键要求是染色质被重塑(即“打开”)，使转录激活蛋白和RNA聚合酶能够访问DNA，从而允许转录复合体的组装，然后将基因转录成信使RNA。该方法利用在人类、酵母、蠕虫和苍蝇基因组项目中发现的保守的染色质基因来：(1)在完整的拟南芥基因组序列中识别相似的基因；(2)从玉米中分离相关基因。拟南芥基因的鉴定为玉米基因的分离提供了极大的便利。染色质基因功能的某些测试需要显性突变，因此每个目标染色质基因都会发生显性负突变。最重要的是，将确定所有突变的特征，以确定它们对基因传递、植物生长和发育的影响，以及一系列全面的生化和表观遗传学测试。这些测试包括组蛋白乙酰化、DNA甲基化、突变和突变的过程、沉默的转基因和转座子的重新激活、农杆菌T-DNA整合的效率和核仁优势。此外，还将测试染色质基因产物与GAL4 DNA结合域的融合对具有GAL4 DNA结合位点的报告转基因的影响，以确定候选基因逆转或促进抑制性染色质形成的能力。这些品系将对分离抑制染色质基因活性的其他突变很有价值。这种“向前”的遗传方法对于确定染色质中许多不高度保守或植物特有的有趣的调控成分将是重要的。该奖项将导致大量有用突变的产生和分类，这些突变将促进对植物基因调控的研究，从而加深对植物控制其基因表达的复杂机制的理解。同样重要的是，将建立染色质数据库和网站，促进科学家之间的交流和传播有关植物和其他生物染色质水平控制的信息。了解植物如何控制基因表达对于了解植物如何生长发育以及它们如何响应和适应环境是至关重要的。作物质量和产量的提高继续依赖于应用新的遗传洞察力和工具，如将从该计划中获得的那些。这项研究将对玉米的遗传改良产生直接影响，也将适用于许多其他重要的农作物。成果：编码染色质蛋白的拟南芥基因的T-DNA插入突变：拟南芥生物资源中心拟南芥基因编码染色质蛋白的RNAi突变体：拟南芥生物资源中心玉米基因编码染色质蛋白的RNAi突变体：玉米遗传储备中心RT-PCR和与染色质基因转录本相对应的cDNA序列：GenBank精选有关拟南芥、玉米和水稻基因编码染色质蛋白的信息：www.chromdb.org

This award supports the generation of mutations in genes that control gene expression at the level of chromatin. The overall goal of the project is to identify and functionally analyze most, if not all, of the several hundred genes in maize (corn) and Arabidopsis that contribute to chromatin-level control of gene expression. Maize is the most important agricultural crop in the US, as well as the premier model system for addressing fundamental questions in monocots, particularly cereals. Chromatin is the proteinaceous material that together with DNA comprises chromosomes. A key requirement for the expression of genes in chromosomes is that chromatin be remodeled (i.e., "opened") in such a way that transcriptional activator proteins and RNA polymerases can have access to the DNA, permitting the assembly of a transcription complex which then transcribes the gene into messenger RNA. The approach exploits conserved chromatin genes identified in the human, yeast, worm, and fly genome projects to: (1) identify similar genes in the complete Arabidopsis genome sequence and (2) isolate related genes from maize. Identification of genes in Arabidopsis greatly facilitates the isolation of genes from maize. Certain tests of chromatin gene function require dominant mutations, so dominant negative mutations will be made for each target chromatin gene. Most importantly, all mutations will be characterized to determine their effects on genetic transmission, plant growth and development, and a comprehensive battery of biochemical and epigenetic tests. These tests include histone acetylation, DNA methylation, the processes of epimutation and paramutation, reactivation of silenced transgenes and transposons, the efficiency of Agrobacterium T-DNA integration, and nucleolar dominance. Also, fusions of chromatin gene products to the GAL4 DNA binding domain will be tested for effects on a reporter transgene possessing a GAL4 DNA binding site to determine the ability of candidate genes to reverse or promote the formation of repressive chromatin. These lines will be valuable for isolation of additional mutations that suppress activity of chromatin genes. This "forward" genetic approach will be important to identify the many interesting regulatory components in chromatin that are not highly conserved or are plant specific. This award will result in the generation and classification of a large set of useful mutations that will facilitate investigations of gene regulation in plants, leading to deeper understanding of the complex mechanisms by which plants control the expression of their genes. Equally important, a chromatin database and web site will be created that will facilitate communication among scientists and dissemination of information on chromatin level control in plants and other organisms. Understanding how plants control gene expression is essential for understanding how plants grow and develop and how they respond and adapt to the environment. Quality and yield improvements in crops continue to depend on applying new genetic insights and tools like those to be gained from this program. The research here will have a direct impact on genetic improvement of maize, and will also be applicable to many other important crop plants.Deliverables:T-DNA insertion mutations of Arabidopsis genes encoding chromatin proteins:Arabidopsis Biological Resource CenterRNAi mutants of Arabidopsis genes encoding chromatin proteins: ArabidopsisBiological Resource CenterRNAi mutants of maize genes encoding chromatin proteins: Maize Genetic StockCenter RT-PCR and cDNA sequences corresponding to transcripts from chromatin genes:Genbank Curated information regarding Arabidopsis, maize and rice genes encodingchromatin proteins: www.chromdb.org