DEVELOPMENT OF LARGE DNA METHODS FOR ARABIDOPSIS

这项建议涉及技术的发展， 便于分离和分析植物的大部分 基因组 目前，克隆植物基因的事还谈不上 已知的比遗传图谱定位在技术上更困难。 限制性片段长度多态性（RFLP）图谱 大多数植物的基因组都有很大的缺口， 染色体“行走”技术很难，如果不是不可能的话。 我们 解决这一问题的方法是开发克隆和 将植物DNA的大片段导入植物细胞。 这些研究将涉及“大DNA”方法的发展 用于染色体步移、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将是建立一个低 分辨率物理图谱的拟南芥基因组使用大 克隆的DNA片段。 大型拟南芥基因组文库 制备含DNA的酵母人工染色体 并克隆到酿酒酵母中。 将分析YAC 通过使用轮廓钳位的脉冲场凝胶电泳（PFGE） 均匀电场（CHEF）装置。 重叠的YAC将 利用分子和酵母遗传学方法进行鉴定和关联 方法. 两条无关但同样重要的染色体 将启动步行/连接项目。 我们建议：1） 使用YACs和PFGE连接拟南芥基因组的宏区域 和2）克隆拟南芥的整个着丝粒区， 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.