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: 6138421
• 负责人: MARK A ESTELLE
• 金额: $ 22.67万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 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/6138421
• 关键词: Arabidopsis; Saccharomyces cerevisiae; chromosome walking; fungal genetics; fungal proteins; fusion gene; gene expression; gene mutation; indoleacetate; microinjections; molecular cloning; phase contrast microscopy; phytohormones; 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：（改编自申请人的摘要）：长期目标 这项研究的目的是了解植物细胞的激素调节 强调生长素。 该提案的具体目标是 确定AXR 1家族的生物化学和细胞功能， 拟南芥和酿酒酵母中的蛋白质。 该系列 蛋白质与泛素激活酶（E1）和遗传相关 研究表明，在拟南芥中，AXR 1基因具有重要作用， 在生长素反应中，这表明受调节的蛋白质降解可能 对生长素作用很重要。 遗传学研究还表明， axr 1表型的抑制子（SAR 1）作用于AXR 1的下游。 的 本提案的具体目标是确定：1）AXR 1和SAR 1在以下方面的作用： 细胞生长：由于已知生长素调节细胞分裂和细胞增殖， 延长PI将确定axr 1突变体是否缺乏 细胞分裂、细胞伸长或两者 以及axr 1根的伸长区。 细胞结构将是 通过用相差显微镜检查薄切片来确定。 将使用BrdU确定细胞分裂的模式和速率。 的 还将讨论SAR 1（axr 1的抑制因子）对细胞结构的影响。 测定 axr 1突变体在根毛伸长方面的缺陷将是 使用显微注射技术进行研究。 2)分子表征 AXR 1的主要功能是减轻SAR 1介导的 抑制生长素反应。 将对这一拟议职能进行调查 通过基于图谱的方法克隆SAR 1。 一个旨在恢复 该基因已经鉴定出大量的信息重组体， 这些将被用来定位染色体步移。 一旦 分离的基因将使用标准技术表征。 第三章 其他SAR基因的表征：至少两个其他SAR基因座 已确定，这些（SAR 2和SAR 3）将在 类似于对SAR 1所描述的方式。 将进行基因检测 以确定三个SAR基因座是否在单一途径中起作用。 如果 适当时，PI将继续进行这些分子表征 基因. 4)AXR 1的细胞定位：AXR 1蛋白的序列 表明其定位于细胞的细胞质和/或细胞核 并且已经鉴定了几个假定的核定位序列 在蛋白质中。 为了确定AXR 1是否定位于细胞核， 将N-和C-末端GUS融合体通过颗粒导入植物细胞 轰击和/或原生质体转化。 5)泛素的作用 生长素作用的途径：本PI先前研究的结果表明， 泛素-蛋白酶体途径可能参与生长素反应。 到 研究这种可能性，特异性蛋白酶体抑制剂的作用 对生长素调节的基因表达的影响将被确定。 6)遗传研究 ENR 1和ENR 2在酵母中的表达：已鉴定出与AXR 1相关的两个基因 在酿酒酵母中。 PI已经证明， ENR 1基因是生存所必需的，而ENR 2基因的缺失则不赋予生存能力。 表型 然而，一个合成的致命的相互作用， ern 2D（ENR 2缺失）和cdc 34 -2突变之间的差异表明， ENR 2和CDC 34直接或间接相互作用。 自从CDC 34 基因编码泛素结合酶，这一结果提供了进一步的 AXR 1样蛋白在泛素途径中的作用的证据。 一 计划在酵母中进行许多遗传和分子实验， 研究ENR 1和ENR 2的生化和细胞功能， 将有望为AXR 1的功能和 AXR 1样基因家族。 7)AXR 1与拟南芥E1基因的相互作用及 E2蛋白：与特定目标6中描述的研究平行， PI将检测AXR 1与各种E1和E2蛋白之间的相互作用 从Arabidopsis 8)AXR 1样蛋白的生物化学功能 上述关于生物化学功能的实验结果 AXR 1样蛋白将通过表征以下功能来扩展： 这些蛋白质在体外。