Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola.
Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola.
复制标题
限制性内切酶介导的整合用于产生炭疽病致病突变体。
DOI：
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发表时间：
2000
影响因子：
3.5
通讯作者：
L. Vaillancourt
中科院分区：
文献类型：
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作者：
M. Thon;E. Nuckles;L. Vaillancourt
我们建立了玉米炭疽病菌的限制性内切酶介导的插入突变(REMI)系统。在这份报告中，我们展示了一种基于REMI的突变方法用于识别新的致病基因。使用REMI可以将转化效率提高27倍，而不是单独使用线性化的质粒。对99个转化子进行了Southern分析，其中51%的转化子含有简单的整合，即在基因组中的一个位置整合了一个载体拷贝。所有这些似乎都在一个独特的位置有一个质粒整合。对六个转化子的整合位点进行测序表明，在所有情况下，质粒整合都发生在相应的限制性内切酶识别位点。我们利用体外生物测定从660个转化子中鉴定出两个致病突变体。利用质粒整合位点两侧的基因组DNA在野生型基因组文库中鉴定相应的粘粒。其中一个突变体的致病力在与宇宙体一起转化后恢复。
We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.