DEVELOPMENT OF LARGE DNA METHODS FOR ARABIDOPSIS

这项提议涉及技术的发展，这些技术将 便于分离和分析植物的大片段 基因组。目前，关于植物基因的克隆还没有什么 更多的是已知的遗传图谱定位是技术上的困难。 的当前限制性片段长度多态(RFLP)图谱 大多数平面基因组包含很大的缺口，这使得标准 染色体“行走”技术即使不是不可能，也是困难的。我们的 解决这一问题的途径是开发克隆和 将大片段的植物DNA导入植物细胞。 这些研究将涉及“大DNA”方法的发展。 使用模型进行染色体行走、基因分离和转移 植物系统，拟南芥。 这项工作的一个重要目标是建设一个 利用大分子标记技术绘制拟南芥基因组物理图谱 克隆的DNA片段。大型拟南芥基因组文库 将制备含DNA的酵母人工染色体(YAC) 并克隆到酿酒酵母中。将对YAC进行分析 脉冲场凝胶电泳法(PFGE) 均匀电场(CHEF)装置。重叠的YAC将 利用分子遗传和酵母遗传进行鉴定和连锁 方法：研究方法。两条互不相关但同样重要的染色体 将启动步行/链接项目。我们建议：1) 利用YACS和PFGE连接拟南芥基因组的宏观区域 2)克隆拟南芥着丝粒的全部区域 染色体。

This proposal concerns the development of technologies which will facilitate the isolation and analysis of large segments of a plant genome. At present, the cloning of plant genes about which nothing more is known than genetic map position is technically difficult. Current restriction fragments length polymorphism (RFLP) maps of most plat genomes contain large gaps which makes standard chromosome "walking" techniques difficult, if not impossible. Our approach to this problem is to develop methods for the cloning and introduction into plant cells of large segments of plant DNA. These studies will involve the development of "large DNA" methods for chromosome walking, gene isolation and transfer using a model plant system, Arabidopsis. An important aim of this work will be the construction of a low resolution physical map of the Arabidopsis genome using large segments of cloned DNA. Genomic libraries of large Arabidopsis DNA-containing yeast artificial chromosomes (YACs) will prepared and cloned into Saccharomyces cerevisiae. YACs will be analyzed by pulsed-field gel electrophoresis (PFGE) using contour-clamped homogeneous electric field (CHEF) apparatus. Overlapping YACs will be identified and linked using both molecular and yeast genetic methods. Two unrelated but equally important chromosome walking/linking projects will be initiated. We propose to: 1) link up macro-regions of the Arabidopsis genome using YACs and PFGE and 2) clone the entire centromere region of an Arabidopsis chromosome.