LIGHT-RESPONSIVE GENES

植物利用光作为重要的信号来调节发育和生长 和代谢过程。 该项目的长期目标是 理解这个信号被传送到 细胞核影响特定基因转录的变化。 这些 研究还应提供有关机制的一般信息 植物基因表达 本提案中描述的实验是 设计用于识别和表征光信号的分量 将光感知与转录因子 反应 我们以前已经开发了豌豆rbcS-3A作为模型系统， 对光反应转录的研究。 这个基因编码小的 核酮糖-1，5-二磷酸羧化酶亚基， 光合碳同化;其转录被诱导三十- 五倍于光。 拟议的研究将整合生物化学， 分子和遗传方法，并将基于以下内容 战略：（1）我们以前已经证明了 核因子GT-1在光响应转录调节中的作用。 我们最近从烟草和拟南芥中分离了cDNA克隆 编码GT-1 我们将使用这些序列来表征编码的 proteins. 这些实验将提供有关机制的信息， 其中GT-1介导其作用。 此外，我们将使用GT-1作为 生物化学底物以鉴定光信号的其它组分 转导链 (2)最近定义的核因子GAF-1， rbcS-3A上游的结合将进一步表征。 GAF-1存在 在更大的丰度核提取物制备的光生长， 与适应黑暗的植物相反。 我们建议分离cDNA克隆编码 这一因素，以确定其光- 依赖的DNA结合活性被调节。 (3)拟南芥 携带det-1突变的植物绕过了正常的光照需求， 启动发展进程。 我们将用这些植物来描述 光响应转录机器的特定组分。 将在det-1突变体背景中测定确定的DNA序列元件; 这些实验应该提供关于 特定的序列元件，以光响应和发育- 调控基因表达。 此外，我们将评估GT-1的状况 和GAF-1 DNA结合活性，以确定 遗传损伤会影响这些转录的特定成分， 机械. (4)光信号转导途径的中间体将 通过筛选诱变的拟南芥植物来鉴定， 显示光调节基因的异常表达。 这些研究报告将 使用携带构建体的转基因植物， 启动子和短定义的DNA序列融合到荧光素酶报告基因 基因 这些基因融合的使用将导致鉴定新的 突变体，包括一些影响DNA结合蛋白以及其他 影响光信号传导的其他成分 途径。 这些突变体将进行遗传分析， 突变体表型。 (5)突变基因将被分离出来， 遗传和分子方法的组合，用于分析其 编码蛋白质。 它们也将被用来研究 野生型基因以及它们在转基因植物中表达的影响 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.