A defect of ligD (human lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae

被引：114

作者：

Mizutani, Osamu ^{[1
]}

Kudo, Youhei ^{[1
]}

Saito, Akemi ^{[1
]}

Matsuura, Tomomi ^{[1
]}

Inoue, Hirokazu ^{[2
]}

Abe, Keietsu ^{[3
]}

Gomi, Katsuya ^{[1
]}

机构：

[1] Tohoku Univ, Grad Sch Agr Sci, Dept Bioind Informat & Genom, Lab Bioind Genom,Aoba Ku, Sendai, Miyagi 9818555, Japan

[2] Saitama Univ, Fac Sci, Dept Regulat Biol, Genet Lab,Sakura Ku, Saitama 3388570, Japan

[3] Tohoku Univ, Grad Sch Agr Sci, Enzymol Lab, Aoba Ku, Sendai, Miyagi 9818555, Japan

来源：

FUNGAL GENETICS AND BIOLOGY | 2008年 / 45卷 / 06期

基金：

日本学术振兴会;

关键词：

Aspergillus oryzae; DNA ligase IV (ligD); gene-targeting; homologous recombination; mitogen-activated protein kinase (MAPK);

D O I：

10.1016/j.fgb.2007.12.010

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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.

引用

页码：878 / 889

页数：12

共 51 条

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