水稻染色体的显微分离与克隆

MICRODISSECTION AND MICROCLONING OF RICE CHROMOSOME

对水稻染色体识别、显微分离与切割、PCR体外扩增和微克隆进行了研究 .利用改进的显微切割装置 ,可以在 1 0 0×油镜下进行染色体分离与微切割 ,不仅解决了水稻染色体的识别和显微分离的困难 ,而且可使染色体特定区微切片段缩小到 7Mb级 ;利用 SUP-PCR可使 fg级水稻染色体 DNA扩增到 μg级 .电泳检测结果表明 ,扩增片断在 80～ 60 0 bp左右 ,经与 p UC1 9连接 ,转化大肠杆菌 DH5a,一次分离的单条水稻染色体可以获得 9×1 0 4克隆 ,一个 7Mb级片段可以获得 3× 1 0 4克隆 ,经分析 ,插入片段大小在 80～ 50 0 bp范围 .该方法为直接从水稻单条染色体或染色体特定区 DNA文库中筛选分子标记奠定了基础 .

In this paper the chromosomal identification, microdissection, PCR amplification microcloning have been studied. we isolated and microdissected the 24 rice chromosomes and 5 fragments from chromosome I by improved microdissection apparatus, not only resolving the identification and microdissection of rice chromosome, but also reducing the microdissected fragment to 7Mb range, with SUP PCR (single unique primer PCR) that can obtain μg class from fg elass DNA templates. The gel electrophoresis of PCR products showed that the size of fragments ranging from 80 to 600bp. These single chromosome PCR products cloning in pUC19 could obtain 9×10 4 clones, even a 7Mb fragment also produce 3×10 4 clones. The restriction enzyme digestion demonstrated that the inserted fragments were approximately from 80 to 500bp. These suggest that our novel method is the basis of screening rice chromosome and chromosome specific regions molecular markers, thus enriched the source of rice genome molecular markers.