Abstract
BackgroundThe 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 FindingsHere 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/SignificanceThis 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.”
Highlights
The vast majority of conventional transgenic plants are produced to improve agronomic traits, through genetic transformation of only one transgenic protein
A system combining the Cre/loxP recombination system and two rare-cutter endonucleases was reported for the sequential assembly of multiple genes onto a transformationcompetent artificial chromosome (TAC) vector [11]
Similar results were obtained as with tobacco, we found that all transgenes linked on the T-DNA were coexpressed. (Figure 5B)
Summary
The vast majority of conventional transgenic plants are produced to improve agronomic traits, through genetic transformation of only one transgenic protein. Agrobacterium vectors have been developed, extensively improved and modified to perform various user-friendly applications, and are currently the favored DNA delivery method for plant genetic transformation [2,3] These vectors are operable in plant science and biotechnology, their ability to simultaneously express several target genes from a single plasmid is limited by their basic design [4]. Yeast recombination has since been applied to construct plasmids and yeast artificial chromosomes (YACs), and is considered an ideal tool for sequence-specific assembly of plasmids or even genomes [9,10] These methods still stay at the level of microorganisms; it will be some time before these can be used in plant genetic breeding. A multigene transformation strategy for use in plant molecular biology and plant genetic breeding is needed
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