DEVELOPMENTAL REGULATION OF THE EPSP SYNTHASE GENE

拟议研究的长期目标是了解分子 高等植物发展的基础。 方法是确定 发育调控基因在特定的时间被激活， 特定组织 5-烯醇式莽草酸-3-磷酸合酶基因 （EPSPS）是一个植物必需基因，具有一个良好的启动子 区域和双峰组织特异性表达模式。 EPSPS RNA是 在成熟的花中高水平表达， 像花朵开放一样丰富。 一个cDNA克隆， 特异性针对EPSPS增强子区域的基因已经被分离。 克隆的 因子具有预期的主要调节因子的特征 EPSPS在成熟植物中的表达-其RNA具有组织特异性 表达模式与EPSPS基因非常相似。 因为 EPSPS在幼苗和花的特定组织中表达， EPSPS是研究组合控制的一个有趣的模型系统 植物基因表达的一个重要组成部分。 转基因植物中的顺式元件分析 这表明，结合同源或 异源反式因子可能在调节EPSPS中起重要作用 表情 使用新开发的EPSPS调节的表征 来自植物的体外转录系统与体内结合 表征应该提供对组合代码的洞察， 在植物发育过程中控制基因表达。 具体目标 这项研究的目的是：1）确定负责 通过使用报告基因的发育调节表达 在转基因植物中的表达和在瞬时测定中， 2）表征克隆抗体的结合特异性; 通过测定两种广泛分离的 EPF 1中的锌指基序; 3）表征转录激活 EPF 1与植物体外转录系统的能力; 4）确定 EPF 1基因调控的性质，通过分析基因组 组织特异性表达决定簇因子的克隆; 5） 表征负责幼苗根中表达的因子， 分析细胞核提取物以鉴定与顺式- 控制根皮层表达的元件; 6）定位和确定 通过产生EPF 1抗体， 确定EPF 1蛋白的丰度和定位; 7）表征 EPF 1在体内的转录激活能力，通过引入 克隆的因子编码的过表达和反义构建体 序列导入转基因植物中，分析对EPSPS基因的影响 表达，并确定是否调节因子浓度 导致表型变化; 8）识别其产物相互作用的基因 通过对表型抑制因子进行遗传筛选， 由异常因子表达产生的改变，以鉴定 与该因子相互作用或被该因子激活。

The long term goal of the proposed research is to understand the molecular basis of development in higher plants. The approach is to determine how a developmentally regulated gene is activated at specific times and in specific tissues. The gene for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is an essential plant gene with a well characterized promoter region and a bimodal tissue-specific expression pattern. EPSPS RNA is expressed at high levels in mature flowers with a dramatic increase in RNA abundance as the flower opens. A cDNA clone for a factor that binds specifically to the EPSPS enhancer region has been isolated. The cloned factor has the characteristics expected of the principal regulatory factor of EPSPS expression in mature plants-its RNA has a tissue-specific expression pattern that is very similar to that of the EPSPS gene. Because EPSPS is expressed in specific tissues in seedlings as well as in flowers, EPSPS serves as an interesting model system to study combinatorial control of gene expression in plants. Cis-element analysis in transgenic plants suggests that combinations of cis-elements that bind homologous or heterologous trans-factors may play a central role in regulating EPSPS expression. Characterization of EPSPS regulation using a newly developed in vitro transcription system from plants combined with in vivo characterization should provide insight into the combinatorial code that controls gene expression throughout plant development. The specific aims of the research are to: 1) identify the DNA sequences responsible for developmentally regulated expression through use of reporter gene expression in transgenic plants and in transient assays with microprojectiles; 2) characterize the binding specificity of a cloned factor, EPF1 by determining the binding specificity of two widely separated zinc finger motifs in EPF1; 3) characterize the transcriptional activating ability of EPF1 with a plant in vitro transcription system; 4) determine the nature of the regulation of the EPF1 gene by analyzing the genomic clone of the factor for the determinants of tissue specific expression; 5) characterize the factors responsible for expression in seedling roots by analyzing nuclear extracts to identify factors that interact with the cis- elements controlling root cortex expression; 6) localize and determine the abundance of EPF1 protein in tissues by generating antibodies to EPF1 to define the abundance and localization of EPF1 protein; 7) characterize the transcriptional activating ability of EPF1 in vivo through introduction of overexpression and anti-sense constructs of the cloned factor coding sequences into transgenic plants to analyze the effect on EPSPS gene expression and to determine whether modulation of factor concentration leads to phenotypic changes; 8) identify the genes whose products interact with the factor by employing a genetic screen for suppressors of phenotypic alterations produced by abnormal factor expression to identify genes that interact with the factor or are activated by the factor.