DIFFERENTIAL EXPRESSION OF THE DIVERSE PLANT ACTINS

不同植物肌动蛋白的差异表达

基本信息

批准号：
6046027
负责人：
Richard Brian Meagher
金额：
$ 32.42万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-04-01 至 2003-12-31
项目状态：
已结题

项目摘要

We have been exploring the Hypothesis (number 1) that the ancient and divergent classes of plant genes have been preserved throughout vascular plant evolution because they have unique patterns of regulation and/or encode proteins with distinct functions. We made use of the compact genome, rapid life cycle, small size, ease of transformation and regeneration, and simple genetics of Arabidopsis to dissect different parts of this hypothesis. Based on the analysis of actin promoter/reporter fusions in hundreds of transgenic plants, RNA levels, and protein levels assayed with isovariant-specific monoclonal antibodies we determined that the three vegetative actins and five reproductive actins each have strong and distinct spatial and temporal expression patterns. These range from constitutive expression of ACT2 in most vegetative tissues, to the phytohormonal induction of ACT7, to the expression of ACT1 in ovules and pollen. The first sequence-based method of isolating plant insertion mutants was developed and used to isolate mutants in four of the eight- expressed actin genes. These mutants have exciting and subtle cell and developmental phenotypes that are revealed when plants are grown under specialized conditions. Multigenerational studies on mutants in three actin genes demonstrated that each gene is under strong selective constraint and is required for the survival of Arabidopsis. The extreme sequence diversity of the plant actin isovariants and their complex expression patterns lead to a second hypothesis. Hypothesis number 2 states that the coexpression of multiple actin isovariants in the same cell results in isovariant dynamics that allow for the temporal and biochemical expansion and buffering of responses of a biological system. A wide variety of molecular, cellular, genetic, and biochemical approaches are needed to address the following Specific Aims: 1) to isolate mutants in each actin and profilin gene; 2) to characterize the phenotypes of these mutants; 3) to determine the spatial or temporal expression patterns of the actin isovariants; 4) to determine the subcellular distribution of coexpressed actins; 5) to dissect the physical/molecular parameters that distinguish the actin isovariants using the functional actin proteins being produced in yeast.

我们一直在探索假设（第1），即在血管植物进化中保存了古代和不同类别的植物基因，因为它们具有具有不同功能的独特调节和/或编码蛋白质的独特模式。我们利用紧凑的基因组，快速生命周期，尺寸较小，易于转化和再生以及拟南芥的简单遗传学来剖析该假设的不同部分。基于对数百种转基因植物中肌动蛋白启动子/报道融合的分析，RNA水平和用异分析特异性的单克隆抗体测定的蛋白质水平，我们确定三种营养肌动蛋白和五种生殖肌动蛋白各有强和不同的空间和时间表达模式。这些范围从大多数植物组织中ACT2的本构表达到ACT7的植物激素诱导，再到ACT1在胚珠和花粉中的表达。开发了基于第一个基于序列的植物插入突变体的方法，并用于在八个表达肌动蛋白基因中的四个中分离突变体。这些突变体具有令人兴奋的细胞和发育表型，当植物在专门条件下生长时会发现。三个肌动蛋白基因突变体的多代化研究表明，每个基因都受到强烈的选择性约束，并且是拟南芥生存所必需的。植物肌动蛋白等异源及其复杂表达模式的极端序列多样性导致了第二个假设。假设2指出，在同一细胞中多种肌动蛋白异源体的共表达导致等异源动力学，从而允许生物系统的时间和生化膨胀和缓冲生物系统的反应。需要多种分子，细胞，遗传和生化方法来解决以下特定目的：1）分离每个肌动蛋白和叶酸蛋白基因中的突变体； 2）表征这些突变体的表型； 3）确定肌动蛋白异源体的空间或时间表达模式； 4）确定共表达肌动蛋白的亚细胞分布； 5）剖析使用酵母中产生的功能性肌动蛋白蛋白来区分肌动蛋白等异源的物理/分子参数。