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Two dominant selectable markers for genetic manipulation in Neurospora crassa.
Current Genetics ( IF 2.5 ) Pub Date : 2020-03-09 , DOI: 10.1007/s00294-020-01063-1
Lingaonan He 1 , Wei Guo 1 , Jiuqi Li 1 , Yuanhao Meng 1 , Ying Wang 1 , Huiqiang Lou 1 , Qun He 1
Affiliation  

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.



中文翻译:

两个主要的选择性标记,可用于Cryospora crassa中的基因操作。

芥菜神经孢霉是用于分子遗传学,生物化学,生理学和分子细胞生物学研究的优秀模型真菌。随着Neurospora研究的飞速发展,对促进更有效和准确的遗传分析的新工具的需求也很高。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. crassa。其中,我们发现克雷萨猪笼草的菌株对氨基糖苷类抗生素G418和神经丝菌素敏感。1000μg/ mL的G418或50μg/ mL的神经丝菌素足以完全抑制Neurospora的生长。当表达哺乳动物细胞中使用的新霉素磷酸转移酶基因(neo)时,猪笼草显示出对G418的强效抗性。这确立了G418抗性标记作为在猪笼草中使用的主要选择标记。类似地,当诺尔丝菌素乙酰转移酶基因(NAT从)诺尔斯氏链霉菌是通过诱导QA-2启动子中的奎尼酸的存在下(QA),粗糙脉孢菌显示到诺尔丝菌素有效的阻力。当NAT是组成由全长表达或截断从该启动子的版本粗糙脉孢菌CFP基因(NCU02193),或由TRPC的启动子构巢曲霉,生长粗糙脉孢菌在存在神经柔丝菌素的情况下,其与Nat的表达水平成正比。最后,使用这两个标记物从N. crassa基因组中敲除wc-2al-1基因。在这项研究中成功开发了这两个标记,扩展了N. crassa的工具箱,也很可能扩展了其他丝状真菌的工具箱。

更新日期:2020-03-09
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