A Novel Method to Specifically Cleave Chromosome-size Plant DNA

9307162吴，这项研究的目标是开发两种新的方法，将染色体大小的DNA特异地切割成大片段，以便构建每条染色体的连续物理图谱。这些片段将被用作制作没有缝隙的每条染色体的低密度物理图谱的起始材料。这种方法的主要优点是，它将允许裂解和回收比以前发表的三种方法所能实现的更大的DNA分子。已经设计了四个新的切割特异性质粒，其中两个已经构建用于本研究。这些质粒将被用来转化水稻，然后再生得到的转基因植株。将进行定量狭缝杂交和基因组印迹分析，以确定每一株转基因植物中切割特异性质粒的整合副本的拷贝数。将获得100多种转基因植物并对其进行鉴定，每一种植物都有几个整合在不同染色体上的卵裂特异质粒的副本。接下来，来自不同转基因植物的染色体DNA将使用一种新的基于酶的策略进行特异性切割，然后使用脉冲场凝胶电泳和杂交分析来分离DNA。这项拟议的研究的最终结果是产生一个连续的、低密度的物理图谱，在两个标记之间有2到7兆基数(Mb)，至少包含水稻12条染色体中的8条。***

9307162 Wu The goal of this proposed research is to develop two new methods for cleaving chromosome-size DNA specifically into large fragments in order to construct a contiguous physical map of each chromosome. These fragments will be used as starting materials in producing a low-density physical map of each chromosome without gaps. The major advantage of this method is that it will allow for the cleavage and recovery of much larger DNA molecules than can be achieved by the three previously published methods. Four novel cleavage-specific plasmids have been designed and two have been constructed for this research. These plasmids will be used to transform Oryza sativa (rice), followed by regeneration of the resulting transgenic plants. Quantitative slot-blot hybridization and genomic blot analysis will be carried out to determine the copy number of the integrated copy of the cleavage-specific plasmid in each transgenic plant. A collection of over 100 transgenic plants will be obtained and characterized, each with several copies of the cleavage-specific plasmids integrated in a different chromosome. Next, the chromosomal DNA from different transgenic plants will be specifically cleaved using a novel enzyme-based strategy, followed by fractionation of the DNA using pulsed field gel electrophoresis and hybridization analysis. The final result of this proposed research is to produce a contiguous, low-density physical map, with between 2 to 7 megabases (mb) in between two markers, of at least 8 out of the 12 chromosomes in rice. ***