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

为了执行这个项目，我们需要几个水稻基因组元素来将它们用作水稻人工染色体的一部分。首先，我们的目标是分离着丝粒克隆并鉴定着丝粒结构。利用谷物着丝粒特异序列，我们可以分离到RCE1516的候选片段。进一步从基因组噬菌体文库中分离出具有RCE1516多个拷贝的较长克隆。对14kb的克隆测序表明，该克隆含有3个1.9kb的重复单位，其中含有RCE1516。该重复单位被命名为RCEI，通过原位杂交可以特异性地定位于水稻染色体的所有着丝粒区域，用另一条水稻着丝粒特异重复序列RCS2筛选也可以分离到定位在水稻5号染色体着丝粒区域的YAC克隆。这一分析揭示了1)每个重叠群大约有1Mb的条纹。2)RCS2重复单位定位为13kb和15kb的BamHI片段上的两个区块，这两个片段彼此非常接近。3)几乎所有的YAC克隆都具有Rce1的多个拷贝，并且似乎均匀地分布在RCS2的两个块的两侧。4)该区域存在9个以上的基因。接下来，我们开始利用其中一个具有着丝粒功能的候选YAC克隆构建水稻人工染色体。为此，通过插入GUS、GFP、HPT基因和水稻端粒序列重建了YAC载体的右臂和左臂。在酵母细胞中，通过同源重组，用重构的YAC臂改造候选着丝粒YAC。

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.