LIGHT-RESPONSIVE GENES

植物利用光作为一个重要的信号来调节 和新陈代谢过程。这个项目的长期目标是 了解此信号传输到 细胞核影响特定基因转录的变化。这些 研究还应提供有关以下机制的一般信息： 植物基因表达。本提案中描述的实验包括 被设计成识别和表征光信号的分量 将光感受与转录调控联系起来的转导途径 回应。我们之前已经开发了豌豆RBCS-3A作为模型系统 对光反应转录的研究。这个基因编码的是小的 核酮糖-1，5-二磷酸羧基酶的亚基，是核酮糖-1，5-二磷酸羧基酶的关键酶 光合作用碳同化；它的转录被诱导30- 光下五折。拟议的研究将结合生物化学、 分子和遗传方法，并将基于以下内容 战略：(1)我们以前已经展示了 核因子GT-1在光反应转录调控中的作用。 我们最近从烟草和拟南芥中分离到了cdna克隆。 对GT-1进行编码。我们将使用这些序列来表征编码的 蛋白质。这些实验将通过以下方式提供有关机制的信息 GT-1介导其作用。此外，我们将使用GT-1作为 用于识别光信号的进一步成分的生化底物 转导链。(2)一种最近定义的核因子GAF-1，它 RBCS-3A上游的结合将进一步表征。存在GAF-1 在从光生长的AS制备的核提取物中含量更丰富 反对适应黑暗的植物。我们建议分离编码基因的cDNA克隆 这一因素是为了确定它的光-- 依赖的DNA结合活性受到调节。(3)拟南芥 携带DET-1突变的植物会绕过正常的光照需求 启动发展过程。我们将用这些植物来刻画 光反应转录机制的特定组件。 确定的DNA序列元件将在Det-1突变背景下进行分析； 这些实验应该提供关于以下贡献的信息 特定的序列元件对光反应和发育- 调控的基因表达。此外，我们将评估GT-1的状态 和GAF-1 DNA结合活性来确定DET-1植物中的 基因损伤会影响转录的这些特定成分 机械设备。(4)光信号转导途径的中间体 通过筛选诱变的拟南芥植株进行鉴定 显示光调节基因的异常表达。这些研究将是 使用转基因植物进行构建，在这些构建中， 启动子和短定义的DNA序列与荧光素酶报告融合 吉恩。这些基因融合的使用应该导致新的 突变体，包括一些影响DNA结合蛋白的突变体以及其他 影响光信号转导的附加组件 小路。这些突变体将接受遗传分析，以确定 突变表型。(5)突变基因将通过一种 遗传学和分子生物学相结合的方法分析其基因 编码的蛋白质。它们还将被用来研究 野生型基因及其在转基因中表达的影响 植物。

Plants utilize light as an important signal to regulate both developmental and metabolic processes. The long term goal of this project is to understand the mechanisms by which this signal is transmitted to the nucleus to affect changes in the transcription of specific genes. These studies should also provide general information about the mechanisms of plant gene expression. The experiments described in this proposal are designed to identify and characterize components of the light signal transduction pathway that link photoperception with a transcriptional response. We have previously developed pea rbcS-3A as a model system for the study of light-responsive transcription. This gene encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, a key enzyme in photosynthetic carbon assimilation; its transcription is induced thirty- five fold by light. The proposed studies will integrate biochemical, molecular and genetic approaches and will be based on the following strategies: (1) We have previously demonstrated a pivotal role for the nuclear factor GT-1 in the modulation of light-responsive transcription. We have recently isolated cDNA clones from both tobacco and Arabidopsis that encode GT-1. We will use these sequences to characterize the encoded proteins. These experiments will provide information on the mechanisms by which GT-1 mediates its effects. Additionally, we will use GT-1 as a biochemical substrate to identify further components of the light signal transduction chain. (2) A recently defined nuclear factor, GAF-1, that binds upstream of rbcS-3A will be further characterized. GAF-1 is present in greater abundance in nuclear extracts prepared from light-grown as opposed to dark-adapted plants. We propose to isolate cDNA clones encoding this factor in order to determine the mechanism by which its light- dependent DNA binding activity is modulated. (3) Arabidopsis thaliana plants carrying a det-1 mutation bypass the normal requirement for light to initiate developmental processes. We will use these plants to characterize specific components of the light responsive transcriptional machinery. Defined DNA sequence elements will be assayed in a det-1 mutant background; these experiments should provide information about the contribution of specific sequence elements to light-responsive and developmentally- regulated gene expression. Additionally, we will assess the status of GT-1 and GAF-1 DNA binding activities in det-1 plants to determine whether the genetic lesion affects these specific components of the transcriptional machinery. (4) Intermediates of the light-signal transduction pathway will be identified by screening mutagenized Arabidopsis plants for those which show aberrant expression of light-regulated genes. These studies will be done using transgenic plants carrying constructs in which both complex promoters and short defined DNA sequences are fused to luciferase reporter gene. Use of these gene fusions should lead to the identification of novel mutants, including some that effect DNA binding proteins as well as other that affect additional components of the light-signal transduction pathways. These mutants will be subjected to genetic analyses to define the mutant phenotype. (5) The mutant genes will be isolated by a combination of genetic and molecular approaches for analysis of their encoded proteins. They will also be used to study the expression of the wildtype genes as well as the effects of their expression in transgenic plants.