REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

这项提议的长期目标是了解全球 植物基因表达的调控。尤其是我们 研究协调基因表达的机制 属于整合氨基酸生物合成途径。真知灼见 从我们最近的工作中开始出现在这个过程中 谷氨酰胺合成酶(GS)基因家族的实验室研究 天冬酰胺合成酶(AS)。这些关键字综合表达 酶类调节植物体内氮素的吸收和运输 植物。我们的研究表明，GS的个别成员 由于基因家族编码不同的产物，这些产物 在特定的细胞类型中差异表达，特别是 发展中的时代，以及对环境信号的回应。 光诱导叶绿体GS2核基因的表达 并伴随着抑制AS1基因的表达。这个 从光明中的GS2表达切换到黑暗中的AS1表达 是戏剧性的，对氮素经济具有生物学意义 植物。我们建议阐明光的新机制-- 抑制AS1的表达并特别注意其 作为诱导型启动子系统的潜力。几款车型将成为 测试以确定GS2的光激活和光- AS1的抑制是由“抑制物/激活物”蛋白或 由不同的因素决定。GS和AS基因的表达也是协调的 当氮必须从富含氮的来源中动员起来时 新陈代谢下沉。相应地，胞质GS3A和AS1的基因是 发芽过程中和固氮根中的协同诱导 结节。我们建议确定与韧皮部有关的因素- 这些基因的特定的和发育调节的表达。 初步结果表明，许多因素相互作用 GS和AS基因的启动子，并提示其机制 这些基因的综合调控是复杂的和 多面性。拟议中的协调因素的研究 在发育过程中调节GS和AS基因将提供一些 对植物基因表达的全球控制的第一次洞察。 这项提议的具体目的是：1)定义光 参与GS2表达激活的调节元件(LRE) 和/或使用报告器光抑制(NLRE)AS1表达 转基因植物中的基因系统。我们将阐明这部小说。 光抑制基因表达的机制及AS1的开发利用 在植物中作为一个“暗”可诱导的启动子系统。2)确定 “亮”和“暗”提取物中独特结合活性的性质 用GS2和AS1启动子的顺式元件检测到。3)识别 韧皮部特有和发育相关的DNA序列 GS3A和AS1在子叶和氮素调控下的表达 固定结节。4)检测韧皮部特异的DNA结合活性 利用一种新的提纯程序。5)鉴定cdna 编码DNA结合蛋白因子的克隆及其性质的确定 因素：因素交互作用。6)检测DNA结合因子的能力 激活或抑制体外转录。7)确定小区- 克隆因子的特异性和体内调控。8)测试模型 异位表达因子(S)对水稻GS和AS的整合调控 含有GS-GUS和AS-GUS转基因植株。9) 确定因子的异位表达是否调节内源性 GS和AS基因的表达和/或影响氮素同化。

The long-term objectives of this proposal ar to understand global control of gene expression in plants. In particular we are investigating the mechanisms which coordinate the expression of genes belonging to integrated amino acid biosynthetic pathways. Insights into this process are beginning to emerge from recent work in our laboratory on the gene families for glutamine synthetase (GS) and asparagine synthetase (AS). The integrated expression of these key enzymes serves to regulate nitrogen assimilation and transport in plants. Our studies have revealed that individual members of the GS and AS gene families encode distinct products which are differentially expressed in particular cell-types, at particular times in development, and in response to environmental signals. Light induces the expression of the nuclear gene for chloroplast GS2 and concomitantly represses the expression of the AS1 gene. The switch from GS2 expression in the light to AS1 expression in the dark is dramatic and has biological significance to nitrogen economy in plants. We propose to elucidate the novel mechanism of light- repressed expression of AS1 with particular attention to its potential as an inducible promoter system. Several models will be tested to determine whether light-activation of GS2 and light- repression of AS1 are mediated by a "repressor/activator" protein or by distinct factors. GS and AS gene expression are also coordinated when nitrogen must be mobilized from nitrogen-rich sources to metabolic sinks. Accordingly, genes for cytosolic GS3A and AS1 are coordinately induced during germination and in nitrogen-fixing root nodules. We propose to identify factors involved in the phloem- specific and developmentally-regulated expression of these genes. Preliminary results suggest that a number of factors interact with the promoters of the GS and AS genes and suggest that the mechanisms underlying the integrated regulation of these genes are complex and multifaceted. The proposed studies on factors which coordinate the regulation of GS and AS genes during development will provide some of the first insights into global control of gene expression in plants. The specific aims of this proposal are to: 1) Define Light Regulatory Elements (LREs) involved in activation of GS2 expression and/or repression (nLRE) of AS1 expression by light using a reporter gene system in transgenic plants. We will elucidate the novel mechanism of light-repressed gene expression and develop AS1 for use as a "dark" inducible promoter system in plants. 2) Determine the nature of unique binding activities in "light" and "dark" extracts detected with cis-elements of the GS2 and AS1 promoters. 3) Identify DNA sequences involved in phloem-specific and developmentally regulated expression of GS3A and AS1 in cotyledons and nitrogen- fixing nodules. 4) Detect phloem-specific DNA binding activities utilizing a novel purification procedure. 5) Characterize cDNA clones encoding DNA binding protein factors and determine the nature of factor:factor interactions. 6) Test ability of DNA-binding factor to activate or repress transcription in vitro. 7) Determine cell- specific and in vivo regulation of cloned factor. 8) Test models for integrated GS and AS regulation by ectopic expression of factor(s) in transgenic plants harboring GS-GUS and AS-GUS transgenes. 9) Determine whether ectopic expression of factor modulates endogenous GS and AS gene expression and/or affects nitrogen assimilation.