Junior Research Group Genetics and Genomics of Fungi, Senckenberg Biodiversity and Climate Research Centre (SBiK-F), Senckenberganlage 25, 60325, Frankfurt am Main, Germany.
Institute of Ecology, Evolution and Diversity, Goethe-University Frankfurt, Max-von-Laue-Str. 13, 60438, Frankfurt am Main, Germany.
Mol Genet Genomics. 2019 Jun;294(3):663-677. doi: 10.1007/s00438-018-01528-6. Epub 2019 Feb 18.
Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (Cbx) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing Cbx as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter P and the heterologous terminator T, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.
杨树菇是许多国家栽培的食用蘑菇，也是研究担子果形成的模式担子菌。为了创建一个易于扩展的定制分子工具集，用于转化和组成型目的基因表达，我们首先创建了一个同源显性标记用于转化体选择。这里使用的基因组测序双核体 A. aegerita AAE-3 的后代单核体被鉴定为对系统性杀菌剂羧菌灵敏感。我们克隆了野生型基因，该基因编码琥珀酸脱氢酶 AaeSdi1 的铁硫蛋白亚基，包括其上下游区域，并引入了单点突变（His237 到 Leu）使其赋予羧菌灵抗性。用来源于孢子或营养单核菌丝体的原生质体进行 PEG 介导的转化，带有所得羧菌灵抗性标记（Cbx）质粒 pSDI1E3，在两种情况下均产生羧菌灵抗性转化体。在选择标记内线性化的质粒 DNA 导致转化体以头到尾重复样的方式异位插入多个质粒 DNA。当使用环形质粒时，有利于异位单整合到真菌基因组中，但在 11 个分析的转化体中的 1 个中也观察到同源位点的基因转换。当使用 Cbx 作为选择标记时，通过 Golden Gate 克隆组装了两个版本的报告基因构建体，其模块易于重组。这些构建体由一个受天然启动子 P 和异源终止子 T 控制的 eGFP 表达盒组成，一次在 eGFP 起始密码子前有一个内含子，一次没有。将任一构建体作为环形质粒 DNA 转化原生质体后，通过 GFP 荧光检测到了 GFP 荧光，这表明在 A. aegerita 中 eGFP 的表达与内含子无关。这为杨树菇的功能遗传学方法铺平了道路，例如通过组成型表达与结实相关的基因。
