高覆盖率水稻ＢＡＣ库的构建及抗病基因相关克隆的筛选

利用含Ｘa4、xa5和xa13 3个水稻白叶枯病抗性基因的累加系ＩＲＢＢ５６构建了一个水稻细菌人工染色体文库，该文库包含５５２９６个克隆，平均插入升段为１３２kb。按水稻基因组为４５０Ｍb计，该文库覆盖１４倍基因组，筛选出任一水稻基因或序列的概率为９９．９９％。用均匀分布的３个叶绿体基因和４个线粒体基因克隆作探针筛选文库，结果显示该文库中含细菌器基因组ＤＮＡ同源序列的克隆数小于１％、用分布于水稻３条不同染色体、分别与Ｘa4、xa5和xa13连锁的ＤＮＡ标记筛选文库，分别检测出１１－１０６个阳性克隆，为克隆这些基因打下了基础。该文库对水稻基因组的高度覆盖率和较大的插入片段，非常适合于物理作图和基因的分离和克隆。

Construction of A Deep Coverage Rice BAC Library and Identification of Clones Associated with Disease-resistant Genes

A BAC library for IRBB56, an accession pyramiding Xa4, xa5 and xa13 three bacterial blight resistance genes, was constructed. The library contains 55 296 clones with an average insert size of 132kb. Based on a haploid genome size of 450Mb, the coverage of the library was about 14 genome equivalents that make it oneof the most comprehensive BAC libraries available in rice and provide 99.99% possibility to isolate any interested rice genes or sequences in the library. To determine the representation of organelle DNA homologues in the library, the library was screened with three different choloroplast genes and four mitochondrial genes, respectively. Results from this screening showed that less than 1% of clones in the library contain organelle genomic DNA homologues. Then, DNA markers on three different chromosomes linked to Xa4, xa5, and xa13, and a PCR fragment of rice UROD gene, were used to screen the library resulting in a range of 11～106 hits that will promote the isolation of these genes. The deep coverage and the large insert size of the library will facilitate physical mapping, isolation, and cloning of rice genes.