Functions of Tubulin and Dynein Genes in Arabidopsis

Snustad 9604491技术拟议研究的长期目标是了解基于微管的细胞骨架组件在植物生长和发育中的作用。植物的形态发生主要由建立细胞分裂平面和细胞伸长轴的细胞骨架过程以及植物细胞特有的微管阵列--前期带和膜质体所控制。在理解这些过程发生的分子机制方面进展甚微。此外，我们仍然对植物中微管相关马达蛋白的结构或功能一无所知。拟议研究的具体目标是首先确定拟南芥17个特征微管蛋白基因中的一些插入突变。这些将被用来研究它们对纯合突变植物表型的影响，并构建和研究携带两个或更多微管蛋白基因插入的多个突变体。第二个目标是开始研究拟南芥中dynein基因家族的复杂性，并对它们进行表征，以达到筛选这些基因插入突变所需的程度。第三个目标是确定动力蛋白重链、中链和轻链基因的插入突变。第四个目标是鉴定拟南芥基因(S)，该基因编码与动物和原生生物8kD动力蛋白轻链同源的蛋白质。利用RNA凝胶印迹杂交实验确定8kD轻链基因(S)的转录积累模式(S)，并利用免疫荧光显微镜对8kD蛋白(S)在植物细胞中的定位，特别是在萌发和伸长的花粉管中。个体目标的进展将取决于插入突变筛查的结果。当一个有趣的突变被发现时，它将成为未来实验的重点。主要目标将是识别单个基因产物的功能，而不是积累所有基因的突变。植物细胞的生长形态和模式是由细胞内的细丝决定的，称为细胞骨架。单个植物细胞的形状和方向对植物形态的形成至关重要。细胞骨架是由几种不同的蛋白质组成的，包括微管蛋白和动力蛋白，它们是作为一个基因家族的产物合成的。目前尚不清楚微管蛋白和动力蛋白基因家族的不同成员是否具有不同的功能角色。在这项研究中，研究人员将分离单个微管蛋白基因被破坏为不起作用的突变体，并用于构建多个微管蛋白基因被破坏的植物。这些实验将导致精确定义哪些微管蛋白基因用于植物发育的特定方面。将对动力蛋白进行平行研究。将对dynein基因家族的大小和多样性进行表征，并构建具有中断的dynein基因的突变体。这项研究很重要，因为它将研究控制植物形状、大小和形态的基本过程之一。***

Snustad 9604491 Technical The long-term goal of the proposed research is to understand the roles of microtubule-based components of the cytoskeleton in plant growth and development. Morphogenesis in plants is controlled primarily by cytoskeletal processes that establish the planes of cell divisions and the axes of cell elongations, and microtubule arrays that are unique to plant cells - preprophase bands and phragmoplasts. Little progress has been made towards understanding the molecular mechanisms by which these processes occur. Moreover, we still know essentially nothing about the structure or function of microtubule-associated motor proteins in plants. The specific objectives of the proposed investigations are to first, identify insertional mutations in some of the 17 characterized tubulin genes of Arabidopsis. These will be used to investigate their effects on the phenotypes of homozygous mutant plants, and to construct and study multiple mutants carrying insertions in two or more tubulin genes. The second objective is to begin to investigate the complexity of the dynein gene families in Arabidopsis and characterize them to the degree needed to screen for insertional mutations in these genes. The third objective is to identify insertional mutations in some of the dynein heavy-, intermediate- and light-chain genes. The fourth objective is to characterize the Arabidopsis gene(s) encoding proteins homologous to the 8 kD dynein light-chains of animals and protists. RNA gel blot hybridization experiments will be used to determine the pattern(s) of transcript accumulation for the 8 kD light-chain gene(s), and immunofluorescence microscopy will be used to localize the 8 kD protein(s) in plant cells, especially in germinating pollen and elongating pollen tubes. The progress of individual objectives will depend on the results of the screens for insertional mutations. When an interesting mutation is identified, it will become the focus of future experiments. The major goal will be to id entify the functions of individual gene products, not to accumulate mutations in all genes. Non Technical The shape and pattern of growth of plant cells is defined by intracellular filaments termed the cytoskeleton. The shape and orientation of individual plant cells is critical to forming the morphology of plants. The cytoskeleton is constructed from several different proteins including tubulin and dynein that are synthesized as products of a gene family. Whether different members of the tubulin and dynein gene families have different functional roles is unknown. In this research the investigators will isolate mutants in which individual genes of tubulin are disrupted to be nonfunctional and used to construct plants in which multiple tubulin genes are disrupted. These experiments will lead to a precise definition of which tubulin genes are used in specific aspects of plant development. Parallel studies will be conducted on dynein. The size and diversity of the dynein gene family will be characterized and mutants with disrupted dynein genes will be constructed. This research is important because it will examine one of the basic processes that controls the shape, size and morphology of plants. ***