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Abstract：
Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.
摘要：
蛹虫草是一种重要的食用菌，可产生多种生物活性化合物。我们先前已经建立了尿苷/尿嘧啶营养缺陷型突变体和相应的根癌农杆菌介导的转化（ATMT）系统，用于使用pyrG作为筛选标记在c中进行遗传表征。在这项研究中,我们构建了一个基于双pyrG和hisB营养缺陷型突变体的ATMT系统。使用尿苷/尿嘧啶营养缺陷型突变体作为背景，pyrG作为选择标记，编码咪唑甘油磷酸脱水酶的hisB基因，组氨酸生物合成所需的，通过同源重组敲除,构建组氨酸营养缺陷型miliaris突变体。然后，删除组氨酸营养缺陷型突变体中的pyrG以构建ΔpyrGΔhisB双重营养缺陷型突变体。Further,我们以GFP和DsRed为报告基因，建立了基于双重营养缺陷型的ATMT转化体系。最后,为了证明这种双重转化系统在基因功能研究中的应用，在双营养缺陷型C中进行了感光基因CmWC-1的敲除和互补。具有组氨酸和尿苷/尿嘧啶营养缺陷型标记的新构建的ATMT系统为药用真菌C.milaris的遗传修饰提供了有希望的工具。
