BACKGROUND The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. RESULTS Here, we present a USER Friendly cloning based technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. CONCLUSION The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.

中文翻译：

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高效四片段克隆构建丝状真菌靶向基因置换载体。

背景全基因组真菌序列信息的快速增加允许对靶基因进行大规模的功能分析。获得定点基因置换、通过启动子置换进行靶向过度表达、帧内表位标记或编码序列与荧光标记（如 GFP）融合的有效转化方法对于该过程至关重要。用于这些实验的载体的构建取决于在选择标记基因的每一侧的两个同源重组序列的定向克隆。结果 在此，我们提出了一种基于用户友好的克隆技术，该技术允许将两个所需的同源重组序列单步克隆到受体载体的不同位点。优点是：实验设计简单，靶序列自由选择，程序少，使用方便。这些载体旨在用于根癌农杆菌和基于原生质体的转化技术。该系统已经通过构建载体进行测试，用于在禾谷镰刀菌中靶向替换17个基因和12个基因的过表达。结果表明，在单个克隆步骤中可以构建四个片段载体，基因替换的平均效率为 84%，靶向过表达的平均效率为 80%。结论 为用户友好克隆设计的新载体提供了一种快速可靠的方法来构建用于真菌靶向基因操作的载体。结果表明，在单个克隆步骤中可以构建四个片段载体，基因替换的平均效率为 84%，靶向过表达的平均效率为 80%。结论 为用户友好克隆设计的新载体提供了一种快速可靠的方法来构建用于真菌靶向基因操作的载体。结果表明，在单个克隆步骤中可以构建四个片段载体，基因替换的平均效率为 84%，靶向过表达的平均效率为 80%。结论 为用户友好克隆设计的新载体为构建用于真菌靶向基因操作的载体提供了一种快速可靠的方法。