DIFFERENTIAL EXPRESSION OF DIVERSE PLANT ACTIN GENES

我们的研究集中在这样一种假设上，即古代的和发散的 植物肌动蛋白基因的分类在维管植物中得到了保存。 进化，因为它们有独特的基因调控和/或模式 编码具有独特功能的蛋白质。在上一次授权期内，我们 对拟南芥的10个成员进行分离、测序和表征 肌动蛋白基因家族(ACT1、2、3、4、5、7、8、9psi、11和12)。他们掉进了 像脊椎动物肌肉一样发散的六个亚纲 肌动蛋白是来自细胞质的肌动蛋白，但植物蛋白的含量要高得多 充电残留物的可变性水平。基于基因特有的诺瑟尔人 RT-PCR分析RNA表达和5‘区/报告融合 在数百种独立的转基因植物中进行了检查，其中8种基因 在5个亚类中，至少在一个细胞中表达水平很高 打字。这五个子类有着截然不同的表达方式 覆盖拟南芥所有组织和发育阶段的模式 (例如，ACT2/ACT8亚类在营养组织中是构成的，而 ACT1/Act3亚类在器官原基和成熟器官中表达 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 包括组成成员(PRF1、2、3)和花粉专一性成员(PRF4)。在……里面 根据这些和其他关于不同的植物肌动蛋白和轮廓蛋白的数据，我们 为我们未来的工作提出第二个假设：有一致的 肌动蛋白和肌动蛋白结合蛋白亚类的表达(如 Profilins)，它们进化出不同的蛋白质-蛋白质相互作用。 基于这两个假设，我们对下一个授权期的具体目标 是：(L)来证明各种肌动蛋白基因或亚家族是 拟南芥正常生长发育所需的特性 缺乏肌动蛋白表达的植物的表型(来自T-DNA 插入文库和通过反义抑制和共抑制)； 确定控制组织特异性表达的主要顺式元件 是否在启动子区域、信使核糖核酸前导或前导内含子；(3)确定 的几个有代表性的成员的详细表达模式 不同的简档亚类(即PRF1和PRF4)；(4)启动 将不同肌动蛋白谱系的起源联系起来的实验 与宏观进化事件有关(例如，具有分生组织起源的ACT1)； 和(5)比较蛋白质-蛋白质相互作用的强度。 肌动蛋白-Profilin对是共表达的，而不是共表达的。这 这项工作将扩大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。

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.