Nishimura M, Nakamura S, Hayashi N, Asakawa S, Shimizu N, Kaku H, Hasebe A, Kawasaki S
National Institute of Agrobiological Resources (NIAR), Japan.
Biosci Biotechnol Biochem. 1998 Aug;62(8):1515-21. doi: 10.1271/bbb.62.1515.
We have constructed a BAC library of the rice blast fungus Magnaporthe grisea consisting of 5760 clones. The insert size ranged from 35 to 175 kbp, with an average of 120 kbp. The library is about 18 genomes equivalent, therefore covering more than 99.999% of the genome. This library is the first to be constructed using a rice pathogenic wild type isolate. Improved high molecular weight DNA size fractionating helped to construct the library with high efficiency. Total library clones were arranged onto two nylon membranes for efficient screening. Test hybridization with a single-copy RFLP marker showed ten positive clones, of which restriction patterns indicated no chimerality or deletions. As a model case of application of this library, the distribution of the well-studied fungal retrotransposons MGSR1, MGR583, and MAGGY and DNA transposons MGR586 and Pot2 was analyzed. Of all the BAC clones, 10%, 13%, 18%, 12%, and 23% contained MGSR1, MGR583, MAGGY, MGR586 and Pot2, respectively. The percentage of clones possessing more than five kinds of transposons was 1.4%, 215 times greater than the expected number. The results show that these transposons were distributed in clusters in the M. grisea genome.
我们构建了一个由5760个克隆组成的稻瘟病菌Magnaporthe grisea的BAC文库。插入片段大小在35至175kbp之间，平均为120kbp。该文库约相当于18个基因组，因此覆盖了超过99.999%的基因组。这个文库是首个使用水稻致病野生型分离株构建的文库。改进的高分子量DNA大小分级有助于高效构建该文库。文库中的所有克隆被排列在两张尼龙膜上以便进行高效筛选。用单拷贝RFLP标记进行的测试杂交显示有10个阳性克隆，其限制酶切图谱表明没有嵌合现象或缺失。作为该文库应用的一个典型案例，分析了研究充分的真菌反转录转座子MGSR1、MGR583和MAGGY以及DNA转座子MGR586和Pot2的分布情况。在所有BAC克隆中，分别有10%、13%、18%、12%和23%含有MGSR1、MGR583、MAGGY、MGR586和Pot2。拥有超过五种转座子的克隆百分比为1.4%，比预期数量大215倍。结果表明这些转座子在稻瘟病菌基因组中呈簇状分布。
