Center for Microbial Biotechnology, Department for Systems Biology, The Technical University of Denmark.
J Microbiol Methods. 2011 Dec;87(3):247-62. doi: 10.1016/j.mimet.2011.09.004. Epub 2011 Sep 17.
Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12 years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient transformation tool for species in which it was previously impossible to conduct molecular genetics experiments. ATMT experiments can be divided into three groups: i) Forward genetics (i.e., random mutagenesis), ii) Reverse genetics (i.e., targeted genome modification and random integration) and iii) the introduction of reporter genes (e.g., GFP, RFP and GUS) that allow in situ monitoring of the fungus. The use of ATMT for forward genetics experiments has primarily included classic random insertional inactivation strategies to obtain loss-of-function mutants. For reverse genetics experiments, ATMT has been used to introduce targeted genome modifications (e.g., disruptions, replacements, overexpression and complementation) and to generate random integrations for complementation, heterologous expression, expression of transcriptional and translational fusion reporters and RNAi-mediated down-regulation of gene expression. This review summarizes the technical advances within the field from 1998 to the summer of 2011, focusing on the development of binary vectors that are compatible with fungal transformation (over 180 general vectors) and methods for constructing binary vectors for targeted integration of T-DNA into fungal genomes.
在过去的 12 年中，根癌农杆菌介导的转化（ATMT）已成为研究各种不同真菌物种的常用技术。发现根癌农杆菌的宿主范围可以扩展到包括真菌，这为以前无法进行分子遗传学实验的物种提供了一种有效的转化工具。ATMT 实验可以分为三组：i）正向遗传学（即随机诱变），ii）反向遗传学（即靶向基因组修饰和随机整合）和 iii）引入报告基因（例如 GFP、RFP 和 GUS），允许原位监测真菌。使用 ATMT 进行正向遗传学实验主要包括经典的随机插入失活策略，以获得功能丧失突变体。对于反向遗传学实验，ATMT 已被用于引入靶向基因组修饰（例如，破坏、替换、过表达和互补），并产生随机整合以进行互补、异源表达、转录和翻译融合报告基因的表达以及 RNAi 介导的基因表达下调。本综述总结了 1998 年至 2011 年夏季期间该领域的技术进展，重点介绍了与真菌转化兼容的二元载体（超过 180 个通用载体）的发展以及用于构建靶向整合 T-DNA 进入真菌基因组的二元载体的方法。
