RMDAP: A Versatile, Ready-To-Use Toolbox for Multigene Genetic Transformation

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.”

Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton

“…The transit peptide of EPSPS gene and Ricin B-chain were ligated with Cry1Ac, generating TP-Cry1Ac-RB and were substantiated by PCR and restriction analysis (Mehlo et al 2005;Steiner et al 2005). The TP-Cry1Ac-RB construct was created after a series of cloning and ligation steps in sub-vectors because of the limitation of the unique restriction sites in T-DNA sequence; an topic which has been explained in detail by Ma et al (2011). After Agrobacteriummediated nuclear transformation, PCR analysis of putative transgenic cotton plants revealed successful integration of desired transgene in the cotton genome ( Figure 9); while Western blot analysis with whole-leaf as well as chloroplast-enriched protein samples displayed single polypeptide of Cry1Ac-RB hybrid protein expressed in chloroplasts ( Figure 10).…”

Engineering crassulacean acid metabolism to improve water-use efficiency

“…Most plant genetic engineering attempts have been limited to the introduction of one or a few genes at a time [123]. To address this limitation, new methods have been developed recently to assemble multigene plant transformation vectors that include a zinc-finger nuclease and homing endonuclease [123], in vivo site-specific assembly [124], recombination-assisted multifunctional DNA assembly [125], or a standardized assembly system based on type IIS restriction enzymes that allows the indefinite expansion of reusable gene modules made from standardized DNA components [126, 127]. The multigene plant transformation vector approach has one major drawback: the maximum number of genes in each vector is limited by the cloning capacity of the recipient vectors.…”

Multigene synergism increases the isoflavone and proanthocyanidin contents of Medicago truncatula

“…To increase the potential for the accumulation of isoflavonoid and/or PA compounds in Medicago , we chose to enhance chalcone biosynthesis by ectopically expressing soya bean CHS and CHI and to reduce the competition between IFS and endogenous F3H using RNA interference. Thus, we designed four plant binary constructs using the Recombination‐assisted Multifunctional DNA Assembly Platform (RMDAP) method (Ma et al ., ). Each vector was designed to generate a distinct synergism between the biosynthesis of isoflavones, flavones and PAs (Figure ).…”

“…To construct the multigene vectors, we employed a versatile multigene assembly system, the Recombination‐assisted Multifunctional DNA Assembly Platform (RMDAP) (Ma et al ., ). The soya bean IFS1 (Jung et al ., ), CHS7 (Akada et al ., ) and CHI1A (Ralston et al ., ) CDSs were amplified from root cDNA, and Xho I (5′ end) and Xba I (3′ end) sites were also incorporated (primers were listed in Table S1).…”