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

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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.