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鉴定和功能分析玉米（玉米）和拟南芥中数百个基因中的大多数，如果不是全部，这些基因有助于染色质水平控制基因表达。玉米是美国最重要的农作物，也是解决单一作物（尤其是谷物）基本问题的首要模式系统。染色质是与DNA一起组成染色体的蛋白质物质。染色体中基因表达的一个关键要求是染色质被重塑（即“打开”），这样转录激活蛋白和RNA聚合酶就可以进入DNA，允许转录复合物的组装，然后将基因转录成信使RNA。该方法利用在人类、酵母、蠕虫和苍蝇基因组计划中发现的保守染色质基因：(1)在拟南芥完整基因组序列中发现相似基因；(2)从玉米中分离相关基因。拟南芥基因的鉴定为从玉米中分离基因提供了便利。染色质基因功能的某些测试需要显性突变，因此每个靶染色质基因将产生显性负突变。最重要的是，所有突变将被表征，以确定其对遗传传递，植物生长和发育的影响，以及生化和表观遗传学测试的综合电池。这些测试包括组蛋白乙酰化、DNA甲基化、表观突变和参数化过程、沉默转基因和转座子的再激活、农杆菌T-DNA整合效率和核仁优势。此外，染色质基因产物与GAL4 DNA结合域的融合将被测试对具有GAL4 DNA结合位点的报告基因的影响，以确定候选基因逆转或促进抑制性染色质形成的能力。这些细胞系将对分离抑制染色质基因活性的其他突变有价值。这种“向前”遗传方法对于识别染色质中许多有趣的调控成分非常重要，这些成分不是高度保守的或植物特异性的。该奖项将导致大量有用突变的产生和分类，这些突变将促进植物基因调控的研究，从而更深入地了解植物控制其基因表达的复杂机制。同样重要的是，将建立一个染色质数据库和网站，以促进科学家之间的交流和传播有关植物和其他生物体中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至关重要。作物的质量和产量的提高继续依赖于应用新的遗传见解和工具，比如从这个项目中获得的那些。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作物。交付成果：拟南芥编码染色质蛋白基因的T-DNA插入突变：拟南芥生物资源中心编码染色质蛋白基因的nai突变：拟南芥生物资源中心编码染色质蛋白基因的nai突变：玉米遗传储备中心染色质基因转录本对应的RT-PCR和cDNA序列：基因库整理的拟南芥、玉米和水稻编码染色质蛋白基因的信息：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