Role of DNA Topology and Chromatin Structure in Phaseolin Gene Regulation

菜豆蛋白（Phaseolin）是菜豆（Phaseolus vulgaris）的主要种子蛋白，在胚胎发生期间高度表达和积累，但在菜豆植物的营养组织中不存在。 在具有β-菜豆蛋白（phas）基因的烟草的稳定转化体中观察到类似的基因表达模式。 与许多植物基因一样，phas基因启动子空间调控所必需的顺式元件位于转录起始位点附近。 虽然许多已知与转录因子相互作用的DNA序列基序存在于该启动子区域中，但这些反式激活因子中没有一个被证明是组织特异性的。 事实上，在通过轰击或电穿孔引入叶细胞的phas/gus构建体的瞬时测定中观察到的大量活性表明营养组织中存在的核因子可以与phas启动子元件有效地相互作用。 相比之下，使用相同构建体的稳定转化的植物即使用一系列生长调节剂处理时，在营养组织中或在来自此类植物的愈伤组织中也不显示表达。 对于一些动物和酵母基因，这些观察结果表明整合到植物染色质中在调节phas基因表达中起作用。 本研究将利用单拷贝phas基因转化烟草，以评估染色质在phas基因表达中的作用。 体内调控phas启动子的顺式元件将通过足迹法来鉴定，足迹法比较营养体（叶）和种子组织中顺式元件与细胞因子的相互作用。 在叶片和种子中定位核小体将确定核小体是否位于phas启动子的特定区域，这可能会阻止反式作用因子的进入，导致营养组织中的失活。本研究的目的是了解phas基因启动子严格的发育和空间调控的细胞机制，特别是确定启动子是如何从营养组织中的完全失活状态转化为胚胎发生过程中的高度活性状态。 菜豆蛋白（Phaseolin）是菜豆（Phaseolus vulgaris）的主要种子蛋白，在胚胎发生过程中高度表达和积累，但在菜豆植物的营养组织中不存在。本研究的目标是了解细胞机制，赋予严格的发育和空间调控的基因编码菜豆蛋白，特别是要确定该基因是如何从一个完全不活跃的状态在营养组织转化为一个高度活跃的状态在胚胎发生。

9604617 Hall Phaseolin, the major seed protein of the bean Phaseolus vulgaris, is highly expressed and accumulated during embryogenesis but is absent from vegetative tissues of bean plants. A similar pattern of gene expression is observed in stable transformants of tobacco with the beta-phaseolin (phas) gene. As for many plant genes, cis-elements essential for spatial regulation of the phas gene promoter lie close to the transcription start site. Although many DNA sequence motifs known to interact with transcription factors are present in this promoter region, none of these transactivators has yet been shown to be tissue specific. Indeed, the substantial activity seen in transient assays of phas/gus constructs introduced into leaf cells by bombardment or electroporation indicates that nuclear factors present in vegetative tissues can interact productively with phas promoter elements. In contrast, stable transformed plants using identical constructs show no expression in vegetative tissues or in calli from such plants even when treated with a range of growth regulators. As for several animal and yeast genes, these observations suggest the integration into plant chromatin plays a role in regulating phas gene expression. This research will use tobacco transformed with a single copy of the phas gene to evaluate the role of chromatin in phas gene expression. Cis-elements that regulate the phas promoter in vivo will be identified by footprinting methods that compare the interaction of cis-elements with cellular factors in vegetative (leaf) and seed tissues. Mapping of nucleosome position in leaves and seeds will determine if nucleosomes are positioned over specific regions of the phas promoter, which could prevent access by transacting factors resulting in the inactivity seen in vegetative tissues. The goals of this research are generally to understand the cellular mechanisms that confer stringent developmental and spatial regulation on the phas gene promoter, and specifically to determine how that promoter is converted from a totally inactive state in vegetative tissues to a highly active state during embryogenesis. Phaseolin, the major seed protein of the bean Phaseolus vulgaris, is highly expressed and accumulated during embryogenesis but is absent from vegetative tissues of bean plants. The goals of this research are generally to understand the cellular mechanisms that confer stringent developmental and spatial regulation on the gene encoding phaseolin, and specifically to determine how that gene is converted from a totally inactive state in vegetative tissues to a highly active state during embryogenesis.