Two dominant selectable markers for genetic manipulation inNeurospora crassa
Two dominant selectable markers for genetic manipulation inNeurospora crassa
复制标题
用于粗糙脉孢菌遗传操作的两个显性选择标记
DOI：
10.1007/s00294-020-01063-1
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发表时间：
2020-08-01
期刊：
影响因子：
2.5
通讯作者：
He, Qun
中科院分区：
文献类型：
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作者：
He, Lingaonan;Guo, Wei;He, Qun
粗糙脉孢菌是分子遗传学、生物化学、生理学和分子细胞生物学研究的理想模式菌。沿着脉孢菌研究的快速进展，对有助于更有效和准确的遗传分析的新工具的需求很高。在这里，我们测试了在酵母中广泛使用的显性选择标记是否适用于N。粗鲁。其中，N. crassa对氨基糖苷类抗生素G418和诺尔丝菌素敏感。1000 μg/mL的G418或50 μg/mL的诺尔丝菌素足以完全抑制脉孢菌生长。当哺乳动物细胞中使用的新霉素磷酸转移酶基因（neo）表达时，N. crassa对G418表现出较强的抗性。这就建立了G418抗性标记作为一个显性选择标记在烟草中的应用。粗鲁。类似地，当诺尔斯链霉菌的诺尔斯菌素乙酰转移酶基因（nat）在奎尼酸（QA）存在下被qa-2启动子诱导时，诺尔斯链霉菌（N. crassa显示出对诺尔丝菌素的强抗性。当nat由来自N. crassa cfp基因（NCU 02193）或构巢曲霉trpC启动子的作用下，N.最后，这两个标记用于敲除来自N. crassa的wc-2或al-1基因。crassa基因组。本研究中这两个标记的成功开发扩展了N. crassa和其他丝状真菌也很可能。
Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 μg/mL of G418 or 50 μg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.