米曲霉RIB40高效同源重组和尿苷/尿嘧啶营养缺陷型菌株的构建

高育青; 张豪杰; 张丹凤; 潘裕添; 张峰

doi:10.13386/j.issn1002-0306.2022040179

米曲霉RIB40高效同源重组和尿苷/尿嘧啶营养缺陷型菌株的构建

Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40

摘要

摘要: 目的：以米曲霉RIB40为出发菌株，构建具有高同源重组效率的营养缺陷型菌株。方法：首先通过同源重组技术和5-氟乳清酸（5-Fluoroorotic Acid，5-FOA）对尿苷/尿嘧啶营养缺陷的筛选作用，获得AopyrG基因缺失的米曲霉RIB40。然后利用烟曲霉AfpyrG基因替换Aoku70得到∆Aoku70敲除菌株，再通过5-FOA的筛选作用使得∆Aoku70菌株基因组发生自身环化，丢失AfpyrG基因。最后为了检验∆Aoku70∆AopyrG菌株的可用性，将编码红色荧光蛋白的mCherry基因敲入至双突变菌株的组蛋白H2B基因上，并通过激光共聚焦显微镜观察红色荧光蛋白质的亚细胞定位。结果：通过AopyrG和Aoku70的双基因敲除，获得了高同源重组效率的营养缺陷型菌株∆Aoku70∆AopyrG。用激光共聚焦观察到红色荧光蛋白定位于细胞核，表明∆Aoku70∆AopyrG菌株可用于米曲霉的基因改造。结论：∆Aoku70∆AopyrG菌株可作为一个高同源重组效率的营养缺陷型出发菌株用于今后米曲霉RIB40的遗传改良。

Abstract: 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.