Development of Molecular Biology Approaches to Arabidopsis Thaliana

本论文拟从拟南芥中克隆、测序和定位转录诱导基因。这些基因将是由硝酸盐诱导的硝酸还原酶基因；由乙烯和病原体诱导的几丁质酶基因；以及赤霉酸诱导的基因。顺式作用位点将通过缺失分析、连接子扫描和插入测试启动子的小片段来定位。为了便于基因分离和定位，将为拟南芥构建一个完整的物理图谱。利用一个横向交替角度为120度的等高线钳制均匀电场，对拟南芥染色体进行物理分离和分离。然后，每个染色体上的NotI片段将被分离出来，并通过与每个RFLP探针杂交来与RFLP遗传图谱相关联。每个片段也将被克隆到新开发的酵母载体中。我们将重点关注拟南芥着丝粒的克隆。这些将用于构建酵母和拟南芥之间的人工染色体穿梭载体。与其他植物相比，拟南芥在分子研究方面具有很大的优势。这项工作的结果将极大地帮助其他利用该植物的研究者，特别是RFLP图谱与NotI片段的整合以及酵母和拟南芥之间人工染色体穿梭载体的开发。

It is proposed to clone, sequence and map transcriptionally inducible genes from Arabidopsis thaliana. These genes will be the nitrate reductase gene inducible by nitrate; chitinase inducible by ethylene and pathogens, and gibberellic acid inducible genes. The cis acting sites will be mapped by deletion analysis, linker scanning and small fragment insertion into a test promoter. In order to facilitate gene isolation and mapping a complete physical map will be constructed for Arabidopsis. The Arabidopsis chromosomes will be physically separated and isolated using an apparatus applying a contour-clamed homogeneous electric field with a transverse alternating angle of 120o. The NotI fragments from each chromosome will then be separated and correlated with the RFLP genetic map by hybridization with each RFLP probe. Each fragment will also be cloned in a newly developed yeast vector. We will focus attention on the cloning of an Arabidopsis centromere. These will be used in the construction of an artificial chromosome shuttle vector between yeast and Arabidopsis. Arabidopsis thaliana has great advantages over other plants for molecular studies. The results of this work will greatly benefit other investigators using this plant, particularly the integration of the RFLP map with NOTI fragments and the development of an artificial chromosome shuttle vector between yeast and Arabidopsis.