Graduate School of Agriculture, Forestry and Fisheries, Kagoshima University, Kagoshima, Japan.
Research Support Center, Institute for Research Promotion, Kagoshima University, Kagoshima, Japan.
Biosci Biotechnol Biochem. 2023 May 19;87(6):672-682. doi: 10.1093/bbb/zbad033.
In this study, we developed an efficient gene targeting system for the osmophilic fungus Aspergillus chevalieri, which is commonly used in the production of a dried bonito, katsuobushi. Specifically, we utilized the clustered regularly interspaced short palindromic repeats/Cas9 system to disrupt the ATP sulfurylase encoding sC gene. This results in methionine auxotroph and selenate-resistance. Additionally, we disrupted the DNA ligase IV encoding ligD gene, which is required for nonhomologous end joining. Using the sC marker and selenate-resistance as a selection pressure, we were able to rescue the sC marker and generate a ΔligD ΔsC strain. We determined that the gene targeting efficiency of the ΔligD ΔsC strain was significantly higher than that of the parental ΔsC strain, which indicates that this strain provides efficient genetic recombination for the genetic analysis of A. chevalieri.
在这项研究中，我们开发了一种针对嗜盐真菌 Aspergillus chevalieri 的高效基因靶向系统，该真菌常用于制作干鲣鱼，即木鱼。具体来说，我们利用簇状规则间隔短回文重复/Cas9 系统来破坏编码 ATP 硫酸化酶的 sC 基因。这会导致蛋氨酸营养缺陷型和硒酸盐抗性。此外，我们还破坏了需要非同源末端连接的 DNA 连接酶 IV 编码 ligD 基因。使用 sC 标记和硒酸盐抗性作为选择压力，我们能够拯救 sC 标记并生成 ΔligD ΔsC 菌株。我们确定，ΔligD ΔsC 菌株的基因靶向效率明显高于亲本 ΔsC 菌株，这表明该菌株为 A. chevalieri 的遗传分析提供了高效的基因重组。
