Yeast recombination-based cloning as an efficient way of constructing vectors for Zymoseptoria tritici.
Yeast recombination-based cloning as an efficient way of constructing vectors for Zymoseptoria tritici.
复制标题
DOI：
10.1016/j.fgb.2015.03.017
复制
发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Steinberg G
中科院分区：
文献类型：
--
作者：
Kilaru S;Steinberg G
基于酵母重组的克隆(YRBC)是一种可靠且廉价的产生质粒的方法。我们为YRBC提供了4个覆盖不同抗性基因的载体。利用这项技术有望迅速产生研究小麦纹枯病菌的分子工具。许多病原真菌具有遗传易感性。分析它们的细胞组织和侵袭机制是毒力决定因素的基础，这得益于利用荧光融合蛋白或条件突变蛋白等位基因等分子工具。这些工具的生成需要高效的克隆方法，因为构建载体通常是一个限速步骤。在这里，我们介绍了一种高效的酵母重组克隆(YRBC)方法来构建真菌ZymosepVictoria tritici的载体。这种方法成本低，避免了在更传统的基于限制/连接的克隆过程中需要的对DNA序列中限制酶位点可用性的依赖。此外，YRBC避免了对目标DNA的修改，事实上，这种潜在的风险限制了特定位点重组系统的使用，如Gateway克隆。相反，在YRBC中，多个DNA片段，具有30个碱基对的重叠序列，被转化到酿酒酵母中，然后同源重组一步就产生了载体。在这里，我们提供了一个详细的实验方案和四个载体，每个载体编码一个不同的显性可选择标记盒，使YRBC的构建能够用于小麦病原菌Z.tritici。
Yeast recombination-based cloning (YRBC) is a reliable and inexpensive way of generating plasmids. We provide 4 vectors for YRBC that a cover different resistance genes. Using this technique promises rapid generation of molecular tools to study Z. tritici. Many pathogenic fungi are genetically tractable. Analysis of their cellular organization and invasion mechanisms underpinning virulence determinants profits from exploiting such molecular tools as fluorescent fusion proteins or conditional mutant protein alleles. Generation of these tools requires efficient cloning methods, as vector construction is often a rate-limiting step. Here, we introduce an efficient yeast recombination-based cloning (YRBC) method to construct vectors for the fungus Zymoseptoria tritici. This method is of low cost and avoids dependency on the availability of restriction enzyme sites in the DNA sequence, as needed in more conventional restriction/ligation-based cloning procedures. Furthermore, YRBC avoids modification of the DNA of interest, indeed this potential risk limits the use of site-specific recombination systems, such as Gateway cloning. Instead, in YRBC, multiple DNA fragments, with 30 bp overlap sequences, are transformed into Saccharomyces cerevisiae, whereupon homologous recombination generates the vector in a single step. Here, we provide a detailed experimental protocol and four vectors, each encoding a different dominant selectable marker cassette, that enable YRBC of constructs to be used in the wheat pathogen Z. tritici.