Chen Xiao-Lin, Yang Jun, Peng You-Liang
State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, Beijing, China.
Methods Mol Biol. 2011;722:213-24. doi: 10.1007/978-1-61779-040-9_16.
With genome sequences of more and more fungi become available, high-throughput systematic -mutagenesis is desirable for functional genomics studies. While a number of random insertional mutagenesis and targeted gene disruption approaches have been used in filamentous fungi, Agrobacterium tumefaciens-mediated Transformation (ATMT) remains one of the most effective methods for identifying genes required for specific fungal developmental or infection processes. Because of its simplicity, ATMT is suitable for large-scale insertion mutagenesis in fungi. Magnaporthe oryzae, the rice blast fungus is a model for studying host-pathogen interactions. Here, we describe protocols for generating a M. oryzae mutant library consisting of over 70,000 ATMT transformants and for identifying genes -disrupted by T-DNA in the mutants by TAIL-PCR.
随着越来越多真菌的基因组序列可得，高通量系统诱变对于功能基因组学研究而言是很有必要的。虽然丝状真菌中已经使用了多种随机插入诱变和靶向基因破坏方法，但根癌农杆菌介导的转化（ATMT）仍然是鉴定特定真菌发育或感染过程所需基因的最有效方法之一。由于其操作简便，ATMT适用于真菌中的大规模插入诱变。稻瘟病菌Magnaporthe oryzae是研究宿主-病原体相互作用的模型。在此，我们描述了用于构建由超过70,000个ATMT转化体组成的稻瘟病菌突变体文库以及通过热不对称交错PCR（TAIL-PCR）鉴定突变体中被T-DNA破坏的基因的方案。
