农杆菌介导的稻瘟病菌转化及致病缺陷突变体筛选  

Agrobacterium tumefaciens-Mediated Transformation of Magnaporthe grisea and Identification of Pathogenicity Defective Mutant

作　　者：刘朋娟[1] 王政逸[1] 王秋华[1] 李德葆[1] 

机构地区：[1]浙江大学农业与生物技术学院生物技术研究所,浙江杭州310029

出　　处：《中国水稻科学》2006年第3期231-237,共7页Chinese Journal of Rice Science

基　　金：国家自然科学基金资助项目(30270861;30570054)

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。Abstract.. Based on the construction of two binary vectors containing hygromycin B, the use of Agrobacterium tumefa- ciens-mediated transformation as a successful method for insertional mutagenesis in rice blast fungus Magna#orthe grisea was reported. A library including more than 4000 transformants with a high transformation efficiency of over 300 hygromycin B re- sistant transformants per 1 X 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 non- pathogenic 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 im- portant biological process blocked in A1-412 was likely to be due to the insertion of T-DNA and the subsequent disruption of gene function.

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 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]