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首页> 外文期刊>Molecular Plant-Microbe Interactions >Transposon impala, a Novel Tool for Gene Tagging in the Rice Blast Fungus Magnaporthe grisea
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Transposon impala, a Novel Tool for Gene Tagging in the Rice Blast Fungus Magnaporthe grisea

机译：转座子黑斑羚，一种用于稻瘟病菌Magnaporthe grisea的基因标记新工具

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摘要

impala , a Tc1-mariner transposable element from Fusarium oxysporum , was introduced into the rice blast fungus Magnaporthe grisea to develop transposon-based insertional mutagenesis. A construct (pNIL160) containing an autonomous impala copy inserted in the promoter of niaD encoding Aspergillus nidulans nitrate reductase was introduced by transformation into a M. grisea nitrate reductase-deficient mutant. impala excision was monitored by restoration of prototrophy for nitrate. Southern analysis of niaD+ revertants revealed that impala was able to excise and reinsert at new loci in M. grisea . As observed for its host Fusarium oxysporum, impala inserted at a TA site left a typical excision footprint of 5 bp. We have shown that a defective impala copy was inactive in M. grisea , yet it can be activated by a functional impala transposase. A transformant carrying a single copy of pNIL160 was used to generate a collection of 350 revertants. Mutants either altered for their mycelial growth (Rev2) or nonpathogenic (Rev77) were obtained. Complementation of Rev77 with a 3-kb genomic fragment from a wild-type locus was successful, demonstrating the tagging of a pathogenicity gene by impala . This gene, called ORP 1, is essential for penetration of host leaves by M. grisea and has no sequence homology to known genes.
机译：黑斑病，来自尖孢镰刀菌的Tc1-mariner转座元件，被引入稻瘟病菌Magnaporthe grisea中，以发展基于转座子的插入诱变。通过转化到稻瘟病菌硝酸还原酶缺陷型突变体中，引入含有插入编码niadugillus nidulans硝酸还原酶的niaD启动子中的自主黑斑羚拷贝的构建体（pNIL160）。通过恢复原营养的硝酸盐监测黑斑羚的切除。对niaD + 回复株的Southern分析表明，黑斑羚能够在稻瘟病菌的新位点切除并重新插入。正如其寄主镰刀菌所观察到的，在TA位点插入的黑斑羚留下的典型切除足迹为5 bp。我们已经表明，有缺陷的黑斑羚复制品在稻瘟病菌中没有活性，但可以被功能性黑斑病转座酶激活。携带单拷贝的pNIL160的转化子用于生成350个回复子的集合。获得了因菌丝体生长（Rev2）或非致病性（Rev77）而改变的突变体。 Rev77与来自野生型基因座的3kb基因组片段成功互补，证明黑斑病可致病性基因的标记。该基因称为ORP 1，对于稻瘟病菌穿透宿主叶至关重要，并且与已知基因没有序列同源性。

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