米曲霉中一个大染色体片段的靶向串联重复
Targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae.
作者信息
Takahashi Tadashi, Sato Atsushi, Ogawa Masahiro, Hanya Yoshiki, Oguma Tetsuya
出版信息
Appl Environ Microbiol. 2014 Aug;80(15):4547-58. doi: 10.1128/AEM.00300-14.
We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5'-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3'-deleted pyrG downstream of the target region. Consequently,strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks(DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.
我们在此描述了首次成功构建米曲霉中一个大染色体片段的靶向串联重复。通过使用在用于串联染色体重复的靶区域上游有一个5'-缺失的pyrG且在靶区域下游有一个3'-缺失的pyrG的菌株，实现了靶向串联染色体重复。因此，成功构建了在8号染色体着丝粒区域附近带有一个210-kb靶向串联染色体重复的菌株以及带有2号染色体一个700-kb区域的靶向串联染色体重复的菌株。带有串联染色体重复的菌株可从亲代菌株的再生原生质体中高效获得。然而，染色体重复的产生并不依赖于I-SceI引入的双链断裂（DSB）。这些菌株的染色体重复在非选择性条件下培养五代后仍能稳定维持。在2号染色体700-kb区域带有串联染色体重复的菌株在固态培养中显示出蛋白酶活性大幅增加，这表明大染色体片段的重复可能是一种有用的新育种技术和基因分析方法。
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