DIFFERENTIAL EXPRESSION OF DIVERSE PLANT ACTIN GENES

多种植物肌动蛋白基因的差异表达

• 批准号: 6039284
• 负责人: Richard Brian Meagher
• 金额: $ 8.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6039284
• 关键词: Arabidopsis; actin binding protein; actins; antisense nucleic acid; fusion gene; gene induction /repression; gene mutation; genetic mapping; genetic promoter element; introns; messenger RNA; plant genetics; polymerase chain reaction; protein protein interaction; protein structure function; reporter genes; site directed mutagenesis; structural genes; tissue mosaicism; transposon /insertion element; yeasts

Our research is focused on the hypothesis that the ancient and divergent classes of plant actin genes have been preserved throughout vascular plant evolution because they have unique patterns of gene regulation and/or encode proteins with unique functions. During the last grant period we isolated, sequenced, and characterized the ten members of the Arabidopsis actin gene family (ACT1, 2, 3, 4, 5, 7, 8, 9psi, 11, & 12). They fall into six subclasses which were approximately as divergent as vertebrate muscle actin is from cytoplasmic actin, but the plant proteins had much higher levels of charged residue variability. Based on gene specific Northerns and RT-PCR analysis of RNA expression and 5' region/reporter fusions examined in hundreds of independent transgenic plants, eight of the genes in five subclasses were expressed at very high levels in at least one cell type. These five subclasses had very distinct and different expression patterns covering all the tissues and developmental stages of Arabidopsis (e.g. the ACT2/ACT8 subclass was constitutive in vegetative tissues, while the ACT1/ACT3 subclass was expressed in organ primordia and mature pollen). An initial characterization of Arabidopsis profilin sequences demonstrated the presence of a large and complex gene family which included constitutive (PRF1, 2, 3) and pollen specific members (PRF4). In light of these and other data on the diverse plant actins and profilins we propose a second hypothesis for our future work: there are concordantly expressed subclasses of actins and actin binding proteins (e.g. profilins), which have evolved distinct protein-protein interactions. Based on these two hypotheses our specific aims for the next grant period are: (l) to demonstrate that the various actin genes or subfamilies are required for normal Arabidopsis growth and development by characterizing the phenotype of plants deficient in actin expression (from a T-DNA insertion library and by antisense suppression and co-suppression); (2) To determine if the major cis-elements controlling tissue specific expression are in the promoter region, mRNA leader or leader intron; (3) to determine the detailed expression patterns of a few representative members of the diverse profilin subclasses (i.e. PRF1 and PRF4); (4) to initiate experiments which will connect the origin of the diverse actin lineages with macroevolutionary events (e.g. ACT1 with the origin of meristems); and (5) to compare the strength of protein-protein interaction between actin-profilin pairs that are co-expressed, with those that are not. This work will expand our basic understanding of the eukaryotic cytoskeleton and enhance our ability to manipulate the properties of higher plants.

我们的研究集中在一个假设上， 在维管植物中保存了许多植物肌动蛋白基因 因为它们具有独特的基因调控模式和/或 编码具有独特功能的蛋白质。在上一次拨款期间，我们 分离、测序并鉴定了拟南芥的十个成员， 肌动蛋白基因家族（ACT 1、2、3、4、5、7、8、9 psi、11和12）。它们落入 六个亚类，它们与脊椎动物的肌肉大致相同， 肌动蛋白是来自细胞质肌动蛋白，但植物蛋白质有更高的 带电残基变异性水平。基于基因特异性的北方人 RNA表达和5'区/报告基因融合的RT-PCR分析 在数百个独立的转基因植物中， 在五个亚类中，至少在一个细胞中以非常高的水平表达 类型.这五个亚类具有非常明显和不同的表达 模式涵盖所有组织和发育阶段的拟南芥 (e.g. ACT 2/ACT 8亚类在营养组织中是组成型，而 ACT 1/ACT 3亚类在器官原基中表达并成熟 花粉）。拟南芥profilin序列的初步鉴定 证明了一个庞大而复杂的基因家族的存在， 包括组成型成员（PRF 1、2、3）和花粉特异性成员（PRF 4）。在 根据这些和其他关于不同植物肌动蛋白和profilins的数据， 为我们未来的工作提出第二个假设： 肌动蛋白和肌动蛋白结合蛋白的表达亚类（例如， profilins），其已经进化出不同的蛋白质-蛋白质相互作用。 基于这两个假设，我们下一个资助期的具体目标 （1）证明各种肌动蛋白基因或亚家族是 所需的正常拟南芥生长和发育，通过表征 缺乏肌动蛋白表达（来自T-DNA）植物表型 插入文库和通过反义抑制和共抑制）;（2） 确定控制组织特异性表达的主要顺式元件 位于启动子区、mRNA前导区或前导内含子;（3）确定 一些代表性成员的详细表达模式， 不同的profilin亚类（即PRF 1和PRF 4）;（4）启动 将不同肌动蛋白谱系的起源联系起来的实验 具有宏观进化事件（例如具有分生组织起源的ACT 1）; 和（5）比较蛋白质-蛋白质相互作用的强度 共表达的肌动蛋白-profilin对，以及不共表达的那些。这 这项工作将扩展我们对真核细胞骨架的基本理解 并增强我们操纵高等植物特性的能力。