Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing Fungus Clitopilus passeckerianus
Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing Fungus Clitopilus passeckerianus
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DOI：
10.1128/aem.01151-09
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发表时间：
2009-11-15
影响因子：
4.4
通讯作者：
Foster, Gary D.
中科院分区：
文献类型：
--
作者：
Kilaru, Sreedhar;Collins, Catherine M.;Foster, Gary D.
我们描述了一种高效的聚乙二醇介导法和农杆菌介导法转化一种具有重要药用价值的担子菌真菌--Plitopilus passeckerianus，它能产生一种二萜抗生素--截胸菌素。利用基于潮霉素、平阳霉素和卡宾选择的3种显性选择标记体系，研究了聚乙二醇法转化帕赛克氏假丝酵母的可行性。利用聚乙二醇法成功地转化了巴氏拟青霉原生质体，并获得了比平阳霉素或卡辛抗性更高的潮霉素抗性转化子。在双孢菇gpdII启动子控制下，利用农杆菌介导法转化含HPH基因的pBGgHg，获得了潮霉素抗性菌落，并以匀浆的菌丝体和子实体鳃组织为起始材料，获得了较好的转化效果。对转化子的Southern杂交分析表明，转化的DNA在基因组中明显随机整合，在聚乙二醇介导体中以多个拷贝为主，而在农杆菌介导体中为单拷贝。从基因组DNA中扩增出Passeckerianus肌动蛋白和微管蛋白启动子，并成功地驱动绿色荧光蛋白和DsRed在Passeckerianus中表达，但只有当构建的载体含有5‘内含子时，证明内含子的存在是高效表达转基因的先决条件。以绿色荧光蛋白(GFP)为靶基因，研究了利用RNA干扰技术实现基因沉默的可行性。在将GFP反义构建物转化为高荧光菌株后，回收的转化子表现出减弱或检测不到的荧光。Northern blotting证实了这一点，显示目标mRNA水平缺失。这表明基因沉默是一种适合于调控巴氏拟青霉基因表达的工具。这项研究中开发的分子工具应该有助于研究这种具有重要药用价值的物种的基因分离或特征。
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.