An Improved Recombineering Toolset for Plants
An Improved Recombineering Toolset for Plants
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DOI：
10.1105/tpc.19.00431
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发表时间：
2020-01-01
期刊：
影响因子：
11.6
通讯作者：
Alonso, Jose M.
中科院分区：
文献类型：
--
作者：
Brumos, Javier;Zhao, Chengsong;Alonso, Jose M.
基因功能研究通常依赖于感兴趣基因的表达，即转录和翻译的融合，并带有专门的标记。理想情况下，这是在自然染色体环境中完成的，以避免潜在的错误表达伪影。尽管最近基因组编辑技术的进步使得直接修改目标基因在其染色体上的位置成为可能，但经典转基因仍然是大多数基因标记研究中首选的实验方法，因为它具有时间效率和可获得性。我们开发了一种基于重组工程的标签系统，它结合了经典转基因方法的便利性和对使用基因组编辑策略直接进行染色体标签所获得的结果的高度置信度。这些简单、可扩展、可定制的重组工程工具包和协议允许生成各种基因修改。此外，我们开发了一种高效的重组酶介导盒交换系统，以便于将所需序列从细菌人工染色体克隆转移到转化相容的二元载体上，从而扩大了重组工程方法的使用范围，使其超出了拟南芥(Arabiopsis Thaliana)的范围。我们通过构建与62个生长素相关基因相对应的250多个全基因翻译融合和123个拟南芥转基因品系，并分析了14个生长素生物合成基因的翻译报告表达模式，证明了该系统的实用性。
Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing have made it possible to directly modify the target genes in their native chromosomal locations, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the results obtained by direct chromosomal tagging using genome-editing strategies. These simple, scalable, customizable recombineering toolsets and protocols allow a variety of genetic modifications to be generated. In addition, we developed a highly efficient recombinase-mediated cassette exchange system to facilitate the transfer of the desired sequences from a bacterial artificial chromosome clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis (Arabidopsis thaliana). We demonstrated the utility of this system by generating more than 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes and characterizing the translational reporter expression patterns for 14 auxin biosynthesis genes.A scalable recombineering procedure for whole-gene editing in plants via homologous recombination in bacteria was used to tag 62 auxin-related genes in 123 transgenic Arabidopsis lines.