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Construction of the rice artificial chromosome and generation of its trasgenic rice. -towards the application of artificial chromosome to the plant breeding technology

水稻人工染色体的构建及其转基因水稻的产生。

基本信息

批准号：
09460006
负责人：
KURATA Nori
金额：
$ 8.13万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

To perform this project, we needs several rice genome elements to use them as parts of the rice artificial chromosome. Firstly we aimed to isolate centromere clones and characterize centromere structure as well. Using cereal centromere specific sequences, we could isolate candidate fragment of RCE1516. To further isolated a longer clone possessing several copies of the RCE1516 from a genomic phage library. Sequencing of the 14kb clone revealed that the clone had three copies of 1.9kb repetitive unit which contains RCE1516. This repetitive unit, designated RCEI, could specifically localize on all centromeric regions of rice chromosomes by in situ hybridization.Screening with another repetitive sequence RCS2 specific for rice centomere could also isolate a YAC clone landed on the centromeric region of rice chromosome 5. Total of 14 YAC clones collected on the region formed 3 contigs and were atructually analyzed. This analysis revealed 1) Each contig streaches about 1Mb. 2) RCS2 repetitive unit localizes as two blocks on 13kb and 15kb BamHI fragments which were very near each others. 3) Almost YAC clones possesses multiple copies of RCE1 and seems to distribute equally on both sides of the two blocks of RCS2. 4) More than 9 genes are present on the region.Next, we started to construct rice artificial chromosome using one of the candidate YAC clones for centromere function. To do this, right and left arms of the YAC vector were reconstructed by inserting GUS, GFP, HPTgenes and rice telomere sequences. Retrofitting of the candidate centromere YAC with the reconstructed YAC arms by homologous recombination in the yeast cells are now in progress.

为了执行这个项目，我们需要几个水稻基因组元件，将它们用作水稻人工染色体的一部分。首先，我们的目的是分离着丝粒克隆并表征着丝粒结构。使用谷物着丝粒特异性序列，我们可以分离RCE1516的候选片段。进一步从基因组噬菌体文库中分离出具有多个 RCE1516 拷贝的较长克隆。对 14kb 克隆进行测序显示，该克隆具有 3 个 1.9kb 重复单元拷贝，其中包含 RCE1516。这个重复单元被命名为RCEI，通过原位杂交可以特异性定位在水稻染色体的所有着丝粒区域。用另一个水稻着丝粒特异的重复序列RCS2进行筛选，也可以分离出落在水稻5号染色体着丝粒区域的YAC克隆。在该区域收集的总共14个YAC克隆形成3个重叠群并进行了结构分析。该分析显示 1) 每个重叠群延伸约 1Mb。 2) RCS2重复单元定位为13kb和15kb BamHI片段上的两个块，这两个块彼此非常接近。 3）几乎YAC克隆拥有多个RCE1副本，并且似乎均匀分布在两个RCS2块的两侧。 4）该区域存在超过9个基因。接下来，我们开始使用候选YAC克隆之一来构建水稻人工染色体，以实现着丝粒功能。为此，通过插入 GUS、GFP、HPT 基因和水稻端粒序列来重建 YAC 载体的右臂和左臂。目前正在酵母细胞中通过同源重组用重建的 YAC 臂改造候选着丝粒 YAC。