An extensible vector toolkit and parts library for advanced engineering of plant genomes
An extensible vector toolkit and parts library for advanced engineering of plant genomes
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DOI：
10.1002/tpg2.20312
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发表时间：
2023-03-09
期刊：
影响因子：
4.2
通讯作者：
Voytas, Daniel F.
中科院分区：
文献类型：
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作者：
Chamness, James C.;Kumar, Jitesh;Voytas, Daniel F.
植物生物技术在转化和基因组工程技术方面取得了新的进展。然而，在植物细胞中传递和协调表达的一个常见要求，将转化结构的设计和组装置于关键时刻，因为所需的试剂套件变得更加复杂。模块化克隆原则简化了载体设计的某些方面，但许多重要组件仍然不适用于生物技术研究中的快速实施。在这里，我们描述了一个通用的金门克隆工具包，用于构建载体。该工具包底盘与广泛接受的遗传部件植物砖标准兼容，并支持通过与现有工具包相比提高的容量、位置灵活性和可扩展性来组装任意复杂的T-DNA。我们还提供了大量新适应的植物砖文库，包括单子叶和双子叶基因表达的调控元件，以及感兴趣的基因的编码序列，如记者、发育调节因子和位点特异性重组酶。最后，我们使用一系列双荧光素酶分析来测量启动子、终止子以及某些启动子中增强子元件的跨盒相互作用对表达的贡献。综上所述，这些公开可用的克隆资源可以极大地加快植物工程新工具的测试和部署。
Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.