Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs in Transgenic Analysis of Plants

Abstract

We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6ÂHis, FLAG, 3ÂHA, 4ÂMyc, 10ÂMyc, GST, T7, and TAP, were employed. Some vectors carry the 2Â35S-promoter for higher-level expression. The kanamycin-and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.

The role of decapping proteins in the miRNA accumulation inArabidopsis thaliana

“…We amplified a DNA fragment containing the DCP2 promoter region (2,555 bp) and the DCP2 ORF from Col-0 genomic DNA using the caccDCP2 Left border and the caccDCP2 Right border primers. These PCR products were then introduced into pENTR/D-TOPO (Invitrogen) and then transferred into pGWB550 by in vitro recombination, 52 using LR Clonase (Invitrogen) to construct pGWB-DCP1-GFP and pGWB-DCP2-GFP. These constructs were introduced into Agrobacterium tumefaciens strain GV3101 and then transformed into heterozygotes of dcp1-1 and dcp2-1 lines, respectively, by the floral dip method.…”

Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis

“…The plasmid constructs were produced using Gateway Technology (Invitrogen) with the destination vectors pGWB405 (Nakagawa et al, 2007), pGWB560 (AB543177; Nakagawa et al, 2007;Nakamura et al, 2010), pBGWF7 (Karimi et al, 2002;Plant System Biology), and FAST-R7 Plant System Biology). The CLO3 complementary DNA (cDNA) fragment from nucleotide 1, corresponding to A of the start codon ATG, to nucleotide 708 was amplified by PCR using the primer set 59-CACCATGGCAG-GAGAGGCAGAGGCTT-39 and 59-GTCTTGTTTGCGAGAATTGGCCC-39.…”

Two Leucine-Rich Repeat Receptor Kinases Mediate Signaling, Linking Cell Wall Biosynthesis and ACC Synthase in Arabidopsis

“…The resultant entry plasmid was used in an LR reaction (as described by the manufacturer; Invitrogen) to introduce the respective genes into the binary pGWB16 (Nakagawa et al, 2007) vector for complementation. The kinase domain of FEI1 were amplified from cDNA by RT-PCR using first-strand cDNA generated from wild-type Col RNA and gene-specific primers (FEI1-C2/FEI1-A5; see Supplemental Table 1 online).…”

“…Kinase-deficient versions of FEI1 or FEI2 were obtained by site-directed mutagenesis using primers containing the desired point mutation (FEI1-M2F/FEI1-M2R and FEI2-M2F/FEI2-M2R; see Supplemental Table 1 online). For expression of a GFP fusion protein, a FEI2 genomic fragment (amplified using primers FEI2-S/FEI2-A4; see Supplemental Table 1 online) was cloned into pENTR-TOPO-D (Invitrogen) and then introduced into the binary vector pGWB5 (Nakagawa et al, 2007). For promoter-GUS fusions, genomic fragments comprising 3 kb of 59 flanking DNA of FEI1 or FEI2 were amplified from wild-type genomic DNA (using primers FEI1-PROM-F1/FEI1-PROM-R1 and FEI2-PROM-F2/FEI2-PROM-R2; see Supplemental Table 1 online), cloned into pENTR4 vector, and then introduced into the binary vector pGWB2 (Nakagawa et al, 2007).…”