Signal-Responsive Transcription of Plant Nuclear Genes

9527364阿里亚斯这些研究的目的是更好地了解植物蛋白质编码基因的表达是如何受到环境刺激的调节。 这里要测试的一个中心假设是，在植物中的防御基因的表达是由G盒结合因子的转录活性的磷酸化依赖的变化调制。 除了了解对所有高等真核生物具有普遍重要性的过程外，拟议的研究可能最终导致开发新颖和可持续的作物保护形式。 植物GBF的转录活性响应于防御线索的变化（例如，非宿主细菌物种或生物激发子）将这些因子的cDNA融合到新的DNA结合结构域，并在含有报道基因的植物细胞中表达。 这些嵌合GBF的磷酸化的变化将通过磷酸肽作图来评估。 将响应防御刺激而磷酸化GBF的蛋白激酶进行生化纯化以获得用于克隆GBF-蛋白激酶cDNA的N-末端序列。 将在体内测试克隆的推定激酶磷酸化GBF和在防御信号传导中起作用的能力。 为了鉴定植物GBF依赖性转录的潜在介质，将用标记的GBF作为探针对植物cDNA表达文库进行远Western筛选。 克隆的蛋白质将通过标准蛋白质结合和转染技术进行功能表征。 高等植物通过合成植物抗毒素来抵抗微生物感染。 在植物抗毒素生物合成中编码酶的“防御”基因的快速和瞬时激活在很大程度上决定了这种反应的时间和幅度。 最近的证据表明，蛋白激酶耦合防御信号的感知增强基因表达。 例如，蛋白激酶的抑制剂阻断植物防御基因的表达，而在其他后生动物系统中刺激蛋白激酶活性的试剂既增强植物防御基因的表达，又导致一些细胞蛋白质的瞬时磷酸化。 植物基因构建因子如何在信号依赖性转录中发挥作用目前尚不清楚，但与动物一样，可能涉及该因子磷酸化状态的变化。 ***

9527364 Arias The goal of these studies is to better understand how the expression of protein-encoding genes of plants are regulated by environmental stimuli. A central hypothesis to be tested here is that defense gene expression in plants is modulated by a phosphorylation-dependent change in the transcriptional activity of G-box binding factors. Aside from understanding a process of general importance to all higher eukaryotes the proposed studies may ultimately lead to the development of novel and sustainable forms of crop protection. Changes in the transcription activity of plant GBF's in response to defense cues (eg., nonhost bacterial species or biotic elicitors) cDNA's of these factors will be fused to a novel DNA-binding domain and expressed in plant cells containing a reporter gene. Changes in the phosphorylation of these chimeric GBF's will be assessed by phosphopeptide mapping. Protein kinases that phosphorylate GBF's in response to defense stimuli will be biochemically purified to obtain N-terminal sequences for cloning GBF-protein kinase cDNA's. Cloned putative kinases will be tested in vivo for their ability to phosphorylate GBF and act in defense-signaling. To identify potential mediators of plant GBF-dependent transcription a far-western screening of plant cDNA expression libraries with labeled GBF as probe will be performed. Cloned proteins will by functionally characterized by standard protein binding and transfection techniques. %%% Higher plants resist microbial infection in part by synthesizing phytoalexins. The rapid and transient activation of "defense" genes that encode for enzymes in phytoalexin biosynthesis largely determines the timing and magnitude of this response. Recent evidence suggests that protein kinases couple defense signal perception to enhanced gene expression. For example, inhibitors of protein kinases block the expression of plant defense genes, whereas agents that stimulate protein kinase activities in other metazoan systems both enhance pla nt defense gene expression and result in the transient phosphorylation of a few cellular proteins. How plant gene building factors function in signal-dependent transcription is currently unknown but, like in animals, may involve changes in the phosphorylation state of the factor. ***