Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstrasse 150, 44801, Bochum, Germany.
Curr Genet. 2021 Apr;67(2):317-330. doi: 10.1007/s00294-020-01143-2. Epub 2020 Dec 24.
The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.
质粒的克隆可能既耗时又昂贵。然而，克隆是许多基因功能分析标准实验的前提，包括缺失突变体的产生和基因产物的定位。在这里，我们提供了 Golden Gate 载体，用于快速、轻松地进行基因融合和基因缺失载体的克隆，适用于多种真菌。在 Golden Gate 克隆中，限制和连接同时在一个反应中发生。我们的载体集包含了常用的 II 型限制性内切酶 BsaI 的识别位点。我们生成了用于 GFP、mRFP 和 3xFLAG 的 C-和 N-末端标记的质粒。对于基因缺失，我们提供了五个不同的供体载体，用于选择标记基因盒。这些包括用于潮霉素 B、诺赛菌素和博莱霉素抗性基因的标准基因盒，以及基于 FLP/FRT 的用于潮霉素 B 和诺赛菌素抗性基因的标记基因盒回收基因盒。为了使克隆变得最可行，我们提供了强大的方案，即（1）本文所述克隆程序的概述，（2）特定的 Golden Gate 反应方案，以及（3）用于质粒克隆和测序以及通过 PCR 和分标记 PCR 生成缺失基因盒的标准引物。我们表明，我们的载体集适用于生物技术相关的青霉和发育模型系统长穗木霉。因此，我们期望这些载体对其他真菌也有益。最后，这些载体可以很容易地适应真菌王国以外的生物。
