FUNCTION OF AXR1 AND AXR1-LIKE GENES IN ARABIDOPSIS

拟南芥中 AXR1 和 AXR1 样基因的功能

• 批准号: 2022364
• 负责人: MARK A ESTELLE
• 金额: $ 20.87万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022364
• 关键词: Arabidopsis; Saccharomyces cerevisiae; chromosome walking; fungal genetics; fusion gene; gene expression; gene mutation; indoleacetate; microinjections; molecular cloning; phase contrast microscopy; plant genetics; plant growth regulators; plant proteins; protein structure function; ubiquitin

DESCRIPTION: (Adapted from the applicant's abstract): The long term goal of the proposed research is to understand hormonal regulation of plant cell growth with emphasis on auxin. The specific goal of this proposal is to determine the biochemical and cellular function of the AXR1 family of proteins in Arabidopsis and Saccharomyces cerevisiae. This family of proteins is related to the ubiquitin-activating enzyme (E1) and genetic studies have shown that, in Arabidopsis, the AXR1 gene has an important role in auxin response suggesting that regulated protein degradation may be important for auxin action. Genetic studies also indicate that one of the suppressors of the axr1 phenotype (SAR1) acts downstream of AXR1. The specific aims of this proposal are to define: 1) Role of AXR1 and SAR1 in Cell Growth: Since auxin is known to regulate both cell division and cell elongation the PI will determine whether the axr1 mutants are deficient in cell division, cell elongation or both by characterizing the cell division and elongation zones of axr1 roots in detail. Cell structure will be determined by examination of thin sections with a phase contrast microscope. The pattern and rate of cell division will be determined using BrdU. The effects of the sar1 (suppressor of axr1) on cell structure will also be determined. The axr1 mutant's deficiency in root hair elongation will be investigated using microinjection techniques. 2) Molecular Characterization of SAR1: The major proposed function for AXR1 is to relieve SAR1-mediated inhibition of auxin response. This proposed function will be investigated by cloning SAR1 using a map-based approach. A project initiated to recover this gene has already identified a large number of informative recombinants in this region and these will be used to orient a chromosome walk. Once isolated, the gene it will be characterized using standard techniques. 3) Characterization of Additional SAR Genes: At least two additional SAR loci have been identified and these (SAR2 and SAR3) will be characterized in a fashion similar to that described for SAR1. Genetic tests will be conducted to determine if the three SAR loci function in a single pathway. If appropriate, the PI will proceed with a molecular characterization of these genes. 4) Cellular Localization of AXR1: The sequence of the AXR1 protein suggests that it is localized to the cytoplasm and/or nucleus of the cell and several putative nuclear localization sequences have been identified within the protein. To determine if AXR1 is localized to the nucleus the PI will introduce N-and C-terminal GUS fusions into plant cells by particle bombardment and/or protoplast transformation. 5) Role of the Ubiquitin Pathway in Auxin Action: The results of this PI's previous studies suggest that the ubiquitin-proteosome pathway may be involved in auxin response. To investigate this possibility, the effects of specific proteosome inhibitors on auxin regulated-gene expression will be determined. 6) Genetic Studies of ENR1 and ENR2 in Yeast: Two genes related to AXR1 have been identified in the yeast Saccharomyces cerevisiae. The PI has demonstrated that the ENR1 gene is essential for viability while deletion of ENR2 confers no phenotype. However, the demonstration that a synthetic lethal interaction between ern2D (deletion of ENR2) and the cdc34-2 mutation indicates that the ENR2 and CDC34 interact, either directly or indirectly. Since the CDC34 gene encodes a ubiquitin-conjugating enzyme, this result provides further evidence for a role for the AXR1-like proteins in the ubiquitin pathway. A number of genetic and molecular experiment are planned in yeast to investigate the biochemical and cellular function of ENR1 and ENR2 and these will hopefully provide important clues to the function of AXR1 and the AXR1-like gene family. 7) Interaction between AXR1 and Arabidopsis E1 and E2 proteins: In parallel with the studies described in Specific Aim 6, the PI will test for interactions between AXR1 and various E1 and E2 proteins from Arabidopsis. 8) Biochemical Function of the AXR1-like Proteins: The results of the experiments described above as to the biochemical function of the AXR1-like proteins will be extended by characterizing the functions of these protein in vitro.

描述：(改编自申请者的摘要)：长期目标 这项拟议的研究的主要目的是了解植物细胞的激素调节 生长，重点是生长素。这项提议的具体目标是 测定AXR1家族的生化和细胞功能 拟南芥和酿酒酵母中的蛋白质。这个家庭 蛋白质与泛素活化酶(E1)和遗传物质有关 研究表明，在拟南芥中，AxR1基因具有重要的作用 生长素反应表明，受调控的蛋白质降解可能是 对生长素的作用很重要。遗传学研究还表明，其中一种 AxR1表型抑制因子(SAR1)作用于AxR1下游。这个 本提案的具体目的是定义：1)AxR1和SAR1在 细胞生长：生长素既能调节细胞分裂又能调节细胞 伸长率等电点将决定AxR1突变体是否缺乏 细胞分裂、细胞伸长或两者兼而有之 以及AxR1根的伸长区。单元格结构将为 通过用相差显微镜检查薄片来确定的。 细胞分裂的模式和速率将使用BrdU来确定。这个 Sar1(AxR1的抑制子)对细胞结构的影响也将是 下定决心。AxR1突变体在根毛伸长方面的缺陷将是 使用微注射技术进行了研究。2)分子表征 关于SAR1：AxR1的主要拟议功能是缓解SAR1介导的 生长素反应的抑制。这一拟议的功能将被调查 通过使用基于地图的方法克隆SAR1。为恢复而启动的项目 该基因已鉴定出大量信息丰富的重组子。 在这个区域，这些将被用来定位染色体行走。一次 分离出来的基因将使用标准技术进行表征。3) 额外SAR基因的特征：至少两个额外的SAR基因座 已经确定，这些(SAR2和SAR3)将在 类似于为SAR1所描述的方式。将进行基因测试 以确定这三个SAR基因座是否在一条路径上起作用。如果 适当时，PI将继续对这些分子进行表征 基因。4)AxR1的细胞定位：AxR1蛋白的序列 提示它定位于细胞质和/或细胞核。 并且已经确定了几个假定的核定位序列 在蛋白质中。要确定AxR1是否定位于细胞核，请使用PI 将N-端和C-端的GUS融合通过粒子引入植物细胞 轰击和/或原生质体转化。5)泛素的作用 生长素作用的途径：这一PI先前的研究结果表明 泛素-蛋白酶体途径可能参与生长素反应。至 研究这种可能性，特定的蛋白酶体抑制剂的作用 关于生长素调节的基因表达将被确定。6)遗传学研究 酵母中的ENR1和ENR2：已鉴定出两个与AxR1相关的基因 在酿酒酵母中。国际和平研究所已经证明 ENR1基因是生存所必需的，而ENR2的缺失则不会 表型。然而，有证据表明，一种人工合成的致命相互作用 在ern2D(ENR2的缺失)和cdc34-2突变之间表明 ENR2和CDC34直接或间接相互作用。自CDC34以来 该基因编码一种泛素结合酶，这一结果进一步提供了 AxR1样蛋白在泛素途径中作用的证据。一个 计划在酵母中进行一些遗传和分子实验，以 研究ENR1和ENR2的生化和细胞功能 将有望为AxR1的功能提供重要线索 AxR1样基因家族。7)AxR1与拟南芥E1的互作和 E2蛋白：与特定目标6中描述的研究同时， PI将测试AxR1与各种E1和E2蛋白之间的相互作用 来自拟南芥。8)类AxR1蛋白的生化功能： 上述关于生物化学功能的实验结果 通过鉴定AXR1样蛋白的功能，可将其扩展 这些蛋白质在体外。