School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, United Kingdom.
Appl Environ Microbiol. 2009 Nov;75(22):7196-204. doi: 10.1128/AEM.01151-09. Epub 2009 Sep 18.
We describe efficient polyethylene glycol (PEG)-mediated and Agrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus, Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation of C. passeckerianus. The PEG-mediated transformation of C. passeckerianus protoplasts was successful and generated hygromycin-resistant transformants more efficiently than either phleomycin or carboxin resistance. Agrobacterium-mediated transformation with plasmid pBGgHg containing hph gene under the control of the Agaricus bisporus gpdII promoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for the Agrobacterium-mediated system within the genome. C. passeckerianus actin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression in C. passeckerianus, but only when constructs contained a 5' intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated using gfp as a target gene easily scored in C. passeckerianus. Upon transformation of gfp antisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression in C. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.
我们描述了一种有效的聚乙二醇（PEG）介导和农杆菌介导的转化系统，用于一种具有药用价值的担子真菌，即 Clitopilus passeckerianus，它产生 pleuromutilin，一种二萜抗生素。我们使用了三种基于潮霉素、弗洛霉素和羧苯磺胺的主要选择标记系统，以研究 PEG 介导的 C. passeckerianus 转化的可行性。PEG 介导的 C. passeckerianus 原生质体转化成功，并且比弗洛霉素或羧苯磺胺抗性更有效地产生潮霉素抗性转化体。使用含有 hph 基因的质粒 pBGgHg 通过 Agaricus bisporus gpdII 启动子进行农杆菌介导的转化，导致潮霉素抗性菌落，并且当使用均质化菌丝体和子实体鳃组织作为起始材料时是成功的。转化体的Southern blot 分析显示，转化 DNA 的随机整合在 PEG 介导的系统中主要是多拷贝，而在农杆菌介导的系统中是单拷贝，在基因组内。从基因组 DNA 扩增出 C. passeckerianus 肌动蛋白和微管蛋白启动子，并成功地在 C. passeckerianus 中驱动绿色荧光蛋白和 DsRed 表达，但只有当构建体包含 5'内含子时才成功，这表明内含子的存在是高效转基因表达的前提。使用 gfp 作为 C. passeckerianus 中易于评分的靶基因，研究了 RNA 干扰介导的基因沉默的可行性。在将 gfp 反义构建体转化为高荧光株系后，回收了显示荧光减少或无法检测到荧光的转化体。这通过 Northern blot 证实，靶 mRNA 水平耗尽。这表明基因沉默是调节 C. passeckerianus 中基因表达的一种合适工具。本研究中开发的分子工具应有助于对该药用重要物种的基因分离或特征进行研究。
