SGER: Development of an In Vitro Chromatin Assembly and Transcription System for Plant Genes

SGER：植物基因体外染色质组装和转录系统的开发

• 批准号: 0139958
• 负责人: Alison Crowe
• 金额: $ 9.18万
• 依托单位: University of Washington
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2003-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0139958&HistoricalAwards=false
• 关键词: SGER; Development; Vitro; Chromatin; Assembly

Studies of both plant and animal genes have illustrated the key role that chromatin and chromatin modulators play in regulating gene expression during development. Correct spatial and developmental expression of phaseolin, the major seed storage protein in bean, requires chromosomal integration. Chromatin structural analyses have established that a nucleosome is rotationally positioned over the phaseolin promoter in non-seed tissue, thereby blocking transcription initiation. This repressive chromatin structure is disrupted prior to beta-phaseolin gene activation in the maturing embryo, suggesting a mechanism by which gene expression can be regulated during development. However, the role that tissue-specific transcription factors and chromatin modulators play in the transition from a silenced to an active phaseolin gene has not been elucidated. The overall aim of this project is to develop an in vitro chromatin assembly and transcription assay to study chromatin-mediated regulation of plant genes. To achieve this goal, the PI will combine existing animal in vitro chromatin assembly systems and a previously developed plant in vitro transcription assay with purified plant transcription factors to reconstitute phaseolin gene regulation in vitro. The three specific aims are 1) to reconstitute physiological chromatin on the phaseolin gene in vitro, 2) to transcribe the phaseolin gene in vitro using a tobacco cell transcription extract, and 3) to reconstitute hormonal and tissue-specific regulation of phaseolin gene expression in vitro. Results from these studies will provide definitive evidence that the chromatin assembly/transcription system faithfully recapitulates the endogenous chromosomal context and tissue-specific expression pattern of a plant gene in vitro. If successful, this in vitro system may provide a universal tool for elucidating the mechanisms driving chromatin-mediated gene regulation in plants. An increased understanding of how chromatin context influences gene expression during plant development will greatly facilitate the engineering of stable transgenic plants for both commercial and agricultural use.

对植物和动物基因的研究已经阐明了染色质和染色质调节剂在发育过程中调控基因表达的关键作用。菜籽素是大豆主要的种子贮藏蛋白，其在空间和发育上的正确表达需要染色体整合。染色质结构分析已经确定，核小体在非种子组织中旋转定位于phaseolin启动子上，从而阻断转录起始。在成熟的胚胎中，这种抑制染色质结构在β -phaseolin基因激活之前被破坏，这表明在发育过程中基因表达可以被调节的机制。然而，组织特异性转录因子和染色质调节剂在沉默相蛋白基因向活性相蛋白基因转变过程中所起的作用尚未阐明。本项目的总体目标是建立一种体外染色质组装和转录试验，以研究染色质介导的植物基因调控。为了实现这一目标，PI将结合现有的动物体外染色质组装系统和先前开发的植物体外转录试验，纯化植物转录因子，在体外重建phaseolin基因调控。三个具体目标是：1)在体外重建phaseolin基因上的生理染色质；2)利用烟草细胞转录提取物在体外转录phaseolin基因；3)在体外重建phaseolin基因表达的激素和组织特异性调控。这些研究的结果将提供明确的证据，证明染色质组装/转录系统忠实地概括了植物基因在体外的内源染色体背景和组织特异性表达模式。如果成功，该体外系统可能为阐明染色质介导的植物基因调控机制提供一个通用工具。加深对植物发育过程中染色质环境如何影响基因表达的理解，将极大地促进商业和农业用途的稳定转基因植物的工程设计。