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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

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【作者】 刘朋娟王政逸王秋华李德葆

【Author】 LIU Peng-juan, WANG Zheng-yi, WANG Qiu-hua, LI De-bao (Biotechnology Institute, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China

【机构】 浙江大学农业与生物技术学院生物技术研究所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浙江杭州310029

【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 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 1×10~ 6 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.

【基金】 国家自然科学基金资助项目(30270861,30570054)
  • 【文献出处】 中国水稻科学 ,Chinese Journal of Rice Science , 编辑部邮箱 ,2006年03期
  • 【分类号】S435.111.4
  • 【被引频次】59
  • 【下载频次】500
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