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Study on molecular breeding by the structural analysis of rice genome.

基于水稻基因组结构分析的分子育种研究

基本信息

批准号：
02454035
负责人：
HIRAI Atsushi
金额：
$ 4.42万
依托单位：
The University of Tokyo (1992)Nagoya University (1990-1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

Techniques for the isolation and fractionation of intact chromosomal DNA have been developed. The isolation of high-molecular-weight (HMW) DNA is performed in agarose blocks and it is then digested for fractionation by pulsed-field gel electrophoresis (PFGE), which can be used to resolve DNAs that are several mega-base pairs in length.Rice is an excellent model system, among crop plants for genome analysis, because of the low degree of complexity of its genome. Well-developed maps of restriction fragment length polymorphisms are available for rice, but methods for handling large pieces of DNA have not been developed that would allow analysis of its genome by PFGE.We have isolated high-molecular-weight DNA of over 5.7 Mb in length from isolated rice germ nuclei. The high-molecular-weight DNA was digested with a number of restriction endonucleases. An ethidium-stained gel after pulsed-field electrophoresis revealed that few of these endonucleases have the ability to cleave rice chromosomal DNA to large fragments of over 200 kb in length, perhaps because of the lower level of methylation of nucleotides in the rice genome than in other genomes. Some restriction endonucleases that do not cut rDNA repeats produced fragments with arrays of rDNA repeats of more than 1.1 Mb, as estimated by Southern analysis. Therefore, it appears that the rice rDNA cluster is constructed with a "physical" succession of more than 130 copies of 8-kb repeating units of rDNA. Genes for alpha-amylase were also detected by Southern method. It indicates our procedure is suitable for analysis of low-copy-number genes.

完整染色体DNA的分离和分级分离技术已经发展起来。高分子量（HMW）DNA的分离是在琼脂糖块中进行的，然后通过脉冲场凝胶电泳（PFGE）进行消化以进行分级分离。脉冲场凝胶电泳（PFGE）可用于解析长度为几个兆碱基对的DNA。成熟的限制性片段长度多态性图谱可用于水稻，但处理大片段DNA的方法尚未开发，这将允许其基因组的PFGE分析。我们已经从分离的水稻胚芽核中分离出长度超过5.7 Mb的高分子量DNA。用多种限制性内切酶消化高分子量DNA。脉冲场电泳后的乙锭染色的凝胶显示，这些内切核酸酶很少有能力切割水稻染色体DNA的长度超过200 kb的大片段，也许是因为在水稻基因组中的核苷酸甲基化水平低于其他基因组。一些不切割rDNA重复序列的限制性内切酶产生的片段具有超过1.1 Mb的rDNA重复序列阵列，如Southern分析所估计的。因此，看来水稻rDNA簇是由超过130个拷贝的8-kb rDNA重复单元的“物理”序列构成的。α-淀粉酶基因也通过Southern方法检测。这表明我们的方法适用于低拷贝数基因的分析。