Laboratory for Conservation and Utilization of Bio-Resources, Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan University, Kunming 650091, China.
Biotechnol Adv. 2013 Dec;31(8):1562-74. doi: 10.1016/j.biotechadv.2013.08.005. Epub 2013 Aug 26.
Advances in genetic transformation techniques have made important contributions to molecular genetics. Various molecular tools and strategies have been developed for functional genomic analysis of filamentous fungi since the first DNA transformation was successfully achieved in Neurospora crassa in 1973. Increasing amounts of genomic data regarding filamentous fungi are continuously reported and large-scale functional studies have become common in a wide range of fungal species. In this review, various molecular tools used in filamentous fungi are compared and discussed, including methods for genetic transformation (e.g., protoplast transformation, electroporation, and microinjection), the construction of random mutant libraries (e.g., restriction enzyme mediated integration, transposon arrayed gene knockout, and Agrobacterium tumefaciens mediated transformation), and the analysis of gene function (e.g., RNA interference and transcription activator-like effector nucleases). We also focused on practical strategies that could enhance the efficiency of genetic manipulation in filamentous fungi, such as choosing a proper screening system and marker genes, assembling target-cassettes or vectors effectively, and transforming into strains that are deficient in the nonhomologous end joining pathway. In summary, we present an up-to-date review on the different molecular tools and latest strategies that have been successfully used in functional genomics in filamentous fungi.
遗传转化技术的进步为分子遗传学做出了重要贡献。自 1973 年首次成功将 DNA 转化到粗糙脉孢菌以来，各种分子工具和策略已被开发出来，用于丝状真菌的功能基因组分析。关于丝状真菌的基因组数据不断增加，并且在许多真菌物种中，大规模的功能研究已经变得很常见。在这篇综述中，比较和讨论了丝状真菌中使用的各种分子工具，包括遗传转化的方法（例如原生质体转化、电穿孔和显微注射）、随机突变文库的构建（例如限制性内切酶介导的整合、转座子阵列基因敲除和根癌农杆菌介导的转化）以及基因功能分析（例如 RNA 干扰和转录激活因子样效应物核酸酶）。我们还重点讨论了可以提高丝状真菌遗传操作效率的实用策略，例如选择适当的筛选系统和标记基因、有效地组装靶盒或载体以及转化到非同源末端连接途径缺陷的菌株中。总之，我们对在丝状真菌功能基因组学中成功使用的不同分子工具和最新策略进行了最新的综述。
