GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.
GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.
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DOI：
10.1371/journal.pone.0083043
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Forner J
中科院分区：
文献类型：
--
作者：
Lampropoulos A;Sutikovic Z;Wenzl C;Maegele I;Lohmann JU;Forner J
构建用于转基因的表达构建体是现代生物学中的基本日常任务之一。传统上，它是基于大量的II型限制性内切酶和T4 DNA连接酶。特别是在需要高通量的长插入和应用的情况下，这种方法受到可用的独特限制性位点的数量以及为每个项目设计单独克隆策略的需要的限制。近年来已经开发了几种替代的克隆系统来克服这些问题，包括基于IIS型酶的金门技术。在这里，我们介绍了我们的GreenGate系统，用于快速组装植物转化结构，这是基于金门方法。GreenGate克隆是简单和有效的，因为它仅使用一种IIS型限制性内切酶，仅依赖于六种类型的插入模块（植物启动子、N-末端标签、编码序列、C-末端标签、植物终止子和植物抗性盒），但同时允许从预克隆的构建块的集合在一个二元目的载体中组装几个表达盒。该系统便宜且可靠，并且当与模块库结合时，大大加快了用于植物转化的克隆和转基因堆叠。
Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.
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