A novel Gateway？-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

In this study, we constructed a novel Gateway？-compatible destination vector, pEG101-SacB/R, by replacing the ccdB gene with a SacB-SacR gene cassette as the negative selectable marker.Our results demonstrate that the new pEG101-SacB/R destination vector can be used for Gateway？ cloning in Agrobacterium tumefaciens. pEG101-SacB/R will be a valuable tool for high-throughput functional analysis of genes in planta.The wide-spread availability of genomic sequences from many organisms has raised interest in characterizing the biological functions of newly discovered genes through various high-throughput methodologies [1]. In order to study gene function, a gene-of-interest needs to be cloned into different plasmid vectors. In plant biology research, the gene-of-interest is frequently cloned into binary vectors that can be used for Agrobacterium-mediated transformation [2].Gateway？ technology was developed as a convenient and fast gene cloning system (Invitrogen？, Carlsbad, CA). This method involves three key steps: (1) amplifying the targeted gene by PCR; (2) directly cloning the PCR product into a TopoEntr/D？ vector without digestion/ligation; and (3) subcloning the targeted gene from the entry vector into any destination vector using the Gateway？ LR cloning technique. The Gateway？ LR cloning reaction is mediated by the site-specific homologous DNA recombination properties of bacteriophage lambda [3]. This method is more convenient than other methods because it does not involve either DNA digestion nor ligation, two processes that can hinder the cloning process [4]. The Gateway？-compatible destination vector harbors a negative selectable marker, the ccdB gene, which produces a toxic protein that is lethal to most E. coli strains, including DH5α [5,6]. During the Gateway？ LR reaction, the ccdB gene in the destination vector is replaced by the targeted gene from an entry vector through site-specific homologous DNA recombination. The LR reaction mixture containing both rec