A defect of ligD (human lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae
A defect of ligD (human lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae
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DOI：
10.1016/j.fgb.2007.12.010
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发表时间：
2008-06-01
影响因子：
3
通讯作者：
Gomi, Katsuya
中科院分区：
文献类型：
--
作者：
Mizutani, Osamu;Kudo, Youhei;Gomi, Katsuya
由于非同源重组在米曲霉中占主导地位，因此在转化过程中很少发生通过同源重组进行的基因靶向。为了开发米曲霉的高效基因靶向系统，我们构建了含有编码人类 DNA 连接酶 IV 同源物的基因 (ligD) 的破坏菌株，该基因参与 DNA 非同源末端连接的最后一步。米曲霉ligD破坏株在营养生长和/或分生孢子形成方面没有表现出明显的缺陷，并且与野生型菌株相比，对导致双链DNA断裂的高浓度甲磺酸甲酯表现出更高的敏感性，但对甲磺酸乙酯和腐草霉素不敏感。 prtR（一种编码调节细胞外蛋白水解基因的转录因子的基因）的基因替换，使用构巢曲霉 sC 基因作为选择性标记，导致 ligD 破坏体的基因靶向效率达到 100%，而当所使用的同源侧翼序列长度超过 0.5 kb 时，野生型的基因靶向效率低于 30%。同样，天冬氨酸蛋白酶编码基因 (pepA) 的基因靶向效率高达 100%。此外，使用米曲霉的ligD破坏系统，我们很容易成功地破坏了五个丝裂原激活蛋白激酶（MAPK）基因，即mpkA、mpkB、hogA、mpkC和米曲霉独特的MAPK（mpkD）。这些结果表明ligD破坏系统对于米曲霉中的基因打靶来说是极其方便的遗传背景。 (c) 2008 Elsevier Inc. 保留所有权利。
Gene-targeting by homologous recombination occurs rarely during transformation since nonhomologous recombination is predominant in Aspergillus oryzae. To develop a highly efficient gene-targeting system for A. oryzae, we constructed disrupted strains harboring a gene (ligD) encoding human DNA ligase IV homolog that is involved in the final step of DNA nonhomologous end joining. The A. oryzae ligD disruptants showed no apparent defect in vegetative growth and/or conidiation, and exhibited increased sensitivity to high concentration of methyl methansulfonate causing double-stranded DNA breaks compared with that of wild-type strain, but not to ethyl methanesulfonate and phleomycin. Gene replacement of the prtR, a gene encoding a transcription factor which regulates extracellular proteolytic genes, using the Aspergillus nidulans sC gene as the selectable marker resulted in 100% of gene-targeting efficiency in the ligD disruptant, compared to less than 30% for a wild-type, when the length of the homologous flanking sequences used was longer than 0.5 kb. Similarly, gene-targeting efficiency was as high as 100% for aspartic protease-encoding gene (pepA). Furthermore, using this ligD disruptant system of A. oryzae, we readily succeeded in disrupting five mitogen-activated protein kinase (MAPK) genes, namely mpkA, mpkB, hogA, mpkC and A. oryzae unique MAPK (mpkD). Such results show that the ligD disruptant system is an extremely convenient genetic background for gene-targeting in A. oryzae. (c) 2008 Elsevier Inc. All rights reserved.