高覆盖率水稻BAC库的构建及抗病基因相关克隆的筛选  

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

作　　者：王文明[1] 江光怀[1] 王世全[1] 朱立煌[1] 翟文学[1] 

机构地区：[1]中国科学院遗传研究所植物生物技术开放实验室,北京100101

出　　处：《Acta Genetica Sinica》2001年第2期102-128,共27页

基　　金：国家自然科学基金!(批准号:39630190);国家973重大基础研究!(批准号:G1998010207);洛克非勒基金!(R

摘　　要：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55 296个克隆，平均插入片段为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胞器基因组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。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.

关 键 词：细菌人工染色体(BAC)文库 基因组 克隆 抗病 水稻 抗病基因 

分 类 号：Q943.2[生物学—植物学]