Signal-Responsive Transcription of Plant Nuclear Genes

这些研究的目的是为了更好地了解植物蛋白编码基因的表达如何受到环境刺激的调节。这里要验证的一个中心假设是，植物中的防御基因表达是由G-box结合因子转录活性的磷酸化依赖性变化调节的。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新的可持续作物保护形式的发展。植物GBF的转录活性对防御信号的响应变化(例如；这些因子的cDNA将融合到一个新的dna结合域，并在含有报告基因的植物细胞中表达。这些嵌合GBF的磷酸化变化将通过磷酸化肽定位来评估。在防御刺激下磷酸化GBF的蛋白激酶将被生化纯化，以获得用于克隆GBF-蛋白激酶cDNA的n端序列。克隆的假定激酶将在体内测试其磷酸化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，将进行以标记的GBF为探针的植物cDNA表达文库的远西部筛选。克隆蛋白将通过标准蛋白结合和转染技术进行功能表征。高等植物抵抗微生物感染的部分途径是合成植物抗毒素。在植物抗菌素生物合成中编码酶的“防御”基因的快速和短暂激活在很大程度上决定了这种反应的时间和强度。最近的证据表明，蛋白激酶将防御信号感知与增强的基因表达结合起来。例如，蛋白激酶抑制剂可以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药物既可以增强植物防御基因的表达，又可以导致一些细胞蛋白的短暂磷酸化。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. ***