Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40
Objective: An auxotrophic strain with high efficiency of homologous recombination was developed in Aspergillus oryzae RIB40. Methods: The AopyrG mutant of A. oryzae RIB40 was firstly obtained using a homologous recombination technique and the 5-fluoroorotic acid (5-FOA) selection for the uridine/ura...
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The editorial department of Science and Technology of Food Industry
2023-01-01
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| Series: | Shipin gongye ke-ji |
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| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022040179 |
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| author | Yuqing GAO Haojie ZHANG Danfeng ZHANG Yutian PAN Feng ZHANG |
| author_facet | Yuqing GAO Haojie ZHANG Danfeng ZHANG Yutian PAN Feng ZHANG |
| author_sort | Yuqing GAO |
| collection | DOAJ |
| description | Objective: An auxotrophic strain with high efficiency of homologous recombination was developed in Aspergillus oryzae RIB40. Methods: The AopyrG mutant of A. oryzae RIB40 was firstly obtained using a homologous recombination technique and the 5-fluoroorotic acid (5-FOA) selection for the uridine/uracil auxotroph. To disrupt the ∆Aoku70 gene, the Aoku70 gene of AopyrG mutant was replaced with A. fumigatus pyrG gene. Then, under the screening effect of 5-FOA, the AfpyrG gene of the ∆Aoku70 strain was deleted via genome self-cyclization. To test the utilization of the resulting strain, the mCherry gene, a red fluorescent protein gene, was integrated with the histone H2B gene in the ∆Aoku70∆AopyrG strain. The subcellular localization of red fluorescent protein was detected by laser confocal microscope. Results: An auxotrophic mutant ∆Aoku70∆AopyrG with high homologous recombination efficiency was established via disruption of the A. oryzae RIB40 genes Aoku70 and AopyrG. The red fluorescence was observed in the nucleus of the fungal cell, indicating the utility of the ∆Aoku70∆AopyrG strain. Conclusion: The ∆Aoku70∆AopyrG strain can be used as a recipient strain with high-efficiency homologous recombination and uridine/uracil auxotroph for future gene modification in A. oryzae RIB40. |
| first_indexed | 2024-04-11T04:38:25Z |
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| language | zho |
| last_indexed | 2024-04-11T04:38:25Z |
| publishDate | 2023-01-01 |
| publisher | The editorial department of Science and Technology of Food Industry |
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| series | Shipin gongye ke-ji |
| spelling | doaj.art-d70079041c1a4f3a82dee9bcbef0d3eb2022-12-28T09:03:10ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062023-01-0144120020710.13386/j.issn1002-0306.20220401792022040179-1Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40Yuqing GAO0Haojie ZHANG1Danfeng ZHANG2Yutian PAN3Feng ZHANG4The Engineering Technological Center of Fungus Active Substances of Fujian Province, Minnan Normal University, Zhangzhou 363000, ChinaCollege of Biological Sciences and Technology, Minnan Normal University, Zhangzhou 363000, ChinaCollege of Biological Sciences and Technology, Minnan Normal University, Zhangzhou 363000, ChinaThe Engineering Technological Center of Fungus Active Substances of Fujian Province, Minnan Normal University, Zhangzhou 363000, ChinaCollege of Biological Sciences and Technology, Minnan Normal University, Zhangzhou 363000, ChinaObjective: An auxotrophic strain with high efficiency of homologous recombination was developed in Aspergillus oryzae RIB40. Methods: The AopyrG mutant of A. oryzae RIB40 was firstly obtained using a homologous recombination technique and the 5-fluoroorotic acid (5-FOA) selection for the uridine/uracil auxotroph. To disrupt the ∆Aoku70 gene, the Aoku70 gene of AopyrG mutant was replaced with A. fumigatus pyrG gene. Then, under the screening effect of 5-FOA, the AfpyrG gene of the ∆Aoku70 strain was deleted via genome self-cyclization. To test the utilization of the resulting strain, the mCherry gene, a red fluorescent protein gene, was integrated with the histone H2B gene in the ∆Aoku70∆AopyrG strain. The subcellular localization of red fluorescent protein was detected by laser confocal microscope. Results: An auxotrophic mutant ∆Aoku70∆AopyrG with high homologous recombination efficiency was established via disruption of the A. oryzae RIB40 genes Aoku70 and AopyrG. The red fluorescence was observed in the nucleus of the fungal cell, indicating the utility of the ∆Aoku70∆AopyrG strain. Conclusion: The ∆Aoku70∆AopyrG strain can be used as a recipient strain with high-efficiency homologous recombination and uridine/uracil auxotroph for future gene modification in A. oryzae RIB40.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022040179aspergillus oryzae rib40uridine/uracil auxotrophpyrgku70fluorescence localization |
| spellingShingle | Yuqing GAO Haojie ZHANG Danfeng ZHANG Yutian PAN Feng ZHANG Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40 Shipin gongye ke-ji aspergillus oryzae rib40 uridine/uracil auxotroph pyrg ku70 fluorescence localization |
| title | Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40 |
| title_full | Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40 |
| title_fullStr | Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40 |
| title_full_unstemmed | Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40 |
| title_short | Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40 |
| title_sort | construction of a high efficiency homologous recombination and uridine uracil auxotroph strain of aspergillus oryzae rib40 |
| topic | aspergillus oryzae rib40 uridine/uracil auxotroph pyrg ku70 fluorescence localization |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022040179 |
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