Abstract: Based on the construction of two binary vectors containing hygromycin B, the use of Agrobacterium tumefaciens-mediated transformation as a successful method for insertional mutagenesis in rice blast fungus Magnaporthe grisea was reported. A library including more than 4000 transformants with a high transformation efficiency of over 300 hygromycin B resistant transformants per 1×10⁶ conidia of M. grisea was generated. All of the hygromycin B resistant transformants tested were mitotically stable after several subcultures onto complete medium without hygromycin. Genomic Southern blot analysis showed that about two thirds of the transformants were single T-DNA insertional events. Through testing pathogenicity of mutational transformants by a rapid barley leaf assay method, an interesting mutant, A1-412, which was completely nonpathogenic to both barley and rice and also lost the ability to undergo infectious growth through abraded leaves was identified. Phenotypic analysis showed that the mutant A1-412 was significantly reduced in conidiation only accounting for 7% conidia of the wild type strain, and was unable to form appressorium on hydrophobic surfaces and its germination was slightly delayed. Southern blot analysis showed that T-DNA inserted into the A1-412 genome was single copy. These data suggest that an important biological process blocked in A1-412 was likely to be due to the insertion of T-DNA and the subsequent disruption of gene function.

Key words: Magnaporthe grisea, Agrobacterium tumefaciens-mediated transformation, insertional mutagenesis, pathogenicity defective mutant

摘要： 在构建了两个含有潮霉素B磷酸转移酶基因双元载体的基础上，成功地实现了农杆菌介导的稻瘟病菌转化，转化效率达每1×106个分生孢子>300个转化子，并得到了4000多个转化子。通过继代培养和PCR检测，证明插入到稻瘟病菌基因组中的潮霉素抗性基因可稳定遗传。Southern杂交分析表明，大约有2/3转化子的TDNA插入是单拷贝的。用大麦叶片离体接种的方法快速测定部分稻瘟病菌转化子的致病性，发现一个致病缺陷突变体：A1412。该突变体不能侵入水稻叶片及擦伤的大麦或水稻叶片，说明A1412突变体在寄主组织中扩展进程被阻断。进一步的表型分析发现A1412突变体的产孢量显著下降，仅为野生菌株的7%，在疏水表面不能形成附着胞，部分分生孢子萌发也略有延迟。Southern 杂交显示A1412基因组中TDNA插入是单拷贝的。上述结果表明，A1412突变体表型的改变可能是由于TDNA插入而使某一具有重要生物学功能的基因失活所致。

关键词: 稻瘟病菌, 根癌农杆菌介导转化, 插入突变, 致病缺陷突变体