State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
PLoS One. 2011;6(5):e19883. doi: 10.1371/journal.pone.0019883. Epub 2011 May 13.
The use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed.
METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-of-principle experiment was confirmed by successfully transferring several heterologous genes into the plant genome.
CONCLUSIONS/SIGNIFICANCE: This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call "one-stop breeding."
利用转基因技术来改良作物的复杂性状，这对多基因转移的现有遗传转化技术提出了挑战。因此，需要一种用于植物分子生物学和植物遗传育种的多基因转化策略。
方法/主要发现：在这里，我们描述了一种通用的、即用型的多基因遗传转化方法，名为重组辅助多功能 DNA 组装平台（RMDAP），它结合了许多现有的植物转化系统的有用特性。该平台整合了三种广泛使用的重组系统，即 Gateway 技术、体内 Cre/loxP 和重组酶，形成了一种高效、可靠的基因组装方法。RMDAP 提出了一种基因堆叠策略，并包含了广泛的灵活、模块化载体，提供了一系列经过功能验证的遗传元件，用于操纵代谢途径中涉及的转基因过表达或基因沉默。特别是构建无标记多基因载体的能力是另一个吸引人的特点。原始载体的内置灵活性大大增加了该系统的可扩展性和适用性。通过成功将几个异源基因转入植物基因组，验证了该原理性实验。
结论/意义：该平台是充分利用代谢途径的协调调控和分子育种潜力的即用型工具包，最终将实现我们所谓的“一站式育种”的目标。
