Ophiocordyceps sinensis, one of the well-known and precious fungal species in the world, parasitizes soil-dwelling larvae of ghost moths on the Tibetan Plateau. The genetic intractability of this extremely psychrophilic and slow-growing O. sinensis fungus is a major limitation on the molecular study. In this study, an Agrobacterium tumefaciens-mediated genetic transformation (ATMT) system for this fungus was established. ATMT procedure was optimized based on the fungal recipient, Agrobacterium strains, and different co-cultivation conditions. Blastospores were ideal recipients for this system. Acetosyringone (AS) was not essential for the transformation of O. sinensis. Sixty to 100 hygromycin B-resistant transformants per 1 × 106 blastospores were obtained. Southern blot analysis indicated the presence of a random single-copy integration of T-DNA into the O. sinensis genome. The insertional transformants with altered growth characters such as colony, blastospore, and fruiting body production were selected to identify the T-DNA flanking sequences by modified hiTAIL-PCR and FPNI-PCR techniques. Eight genes, including genes for an MFS transporter, a 2-oxoglutarate dehydrogenase, a DNA-directed RNA polymerase III complex subunit Rpc37, a cytochrome oxidase subunit I, a mitochondrial import inner membrane translocase subunit tim54, a cytidine deaminase, a phosphoribosylaminoimidazole carboxylase, and a histone H3-like centromeric protein cse-4 were identified. This ATMT system provides a useful tool for gene discovery and characterization of O. sinensis and contributes to the better understanding of the mysterious life cycle of O. sinensis and the molecular interaction between this fungus and its host insects.

中文翻译：

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基于根癌农杆菌介导的转化鉴定与药用真菌中华麦冬（Ophiocordyceps sinensis）生长特性有关的基因。

中华（Ophiocordyceps sinensis）是世界上著名的珍贵真菌之一，寄生于青藏高原的幽灵蛾类的土生幼虫中。这种极富精神性且生长缓慢的中华稻草真菌的遗传难治性是分子研究的主要限制。在这项研究中，建立了这种真菌的根癌农杆菌介导的遗传转化（ATMT）系统。根据真菌受体，农杆菌菌株和不同的共培养条件优化了ATMT程序。芽孢是该系统的理想接受者。乙酰丁香酮（AS）不是转化中华稻草所必需的。每1×106个芽孢中有60至100个潮霉素B抗性转化子。Southern印迹分析表明存在将T-DNA随机单拷贝整合到中华稻草基因组中的现象。通过改良的hiTAIL-PCR和FPNI-PCR技术，选择具有改变的生长特性的插入转化体，例如菌落，芽孢杆菌和子实体产量，以鉴定T-DNA侧翼序列。八个基因，包括MFS转运蛋白，2-氧戊二酸脱氢酶，DNA定向RNA聚合酶III复合亚基Rpc37，细胞色素氧化酶亚基I，线粒体导入内膜转座酶亚基tim54，胞苷脱氨酶，磷酸核糖氨基氨基咪唑羧化酶，鉴定出组蛋白H3样着丝粒蛋白cse-4。该ATMT系统为O的基因发现和表征提供了有用的工具。