A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.
A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.
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DOI：
10.1016/j.fgb.2015.03.018
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发表时间：
2015-06
期刊：
影响因子：
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通讯作者：
Steinberg G
中科院分区：
文献类型：
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作者：
Kilaru S;Schuster M;Latz M;Das Gupta S;Steinberg N;Fones H;Gurr SJ;Talbot NJ;Steinberg G
我们建立了Z.用于构建体作为单拷贝的靶向整合的三个基因座。构建体的整合传递了羧菌素抗性。我们提供了一种载体，用于将eGFP表达构建体整合到sdi 1基因座中。整合到sdi 1基因座上不影响Z.三个月。了解真菌病原体的细胞组织和生物学需要突变等位基因或荧光融合蛋白构建体的基因组整合的准确方法。在Zymoseptoria triumphora中，这可以通过将质粒DNA随机整合到该小麦病原体的基因组中来实现。然而，非靶向异位整合具有不希望的副作用的风险，例如由于靶向调控元件而改变的基因表达，或整合到基因组的蛋白质编码区中后的基因破坏。在这里，我们建立了琥珀酸脱氢酶（sdi1）位点作为一个单一的“软着陆”网站有针对性的异位整合的遗传构建体，通过使用一个抗二氢大麻酚sdi1R等位基因，携带点突变H267L。我们使用各种绿色和红色荧光融合构建体，并显示97%的所有转化体正确整合到sdi 1位点作为单拷贝。我们还证明，这种整合并不影响致病性Z。因此，sdi 1基因座是一个有用的工具，毒力分析在转基因Z。三尖杉菌株此外，我们已经开发了一种载体，其促进酵母重组克隆，从而允许在单个克隆步骤中组装多个重叠DNA片段，用于高通量载体和菌株产生。
We establish the sdi1 of Z. tritici locus for targeted integration of constructs as single copies. Integration of constructs conveys carboxin resistance. We provide a vector for integration of eGFP-expressing construct into the sdi1 locus. Integration into sdi1 locus is not affecting virulence of Z. tritici. Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single “soft-landing” site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1R allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.