Multisite gateway lr recombination reaction

cDNAs encoding full-length mouse TWIK-1 (NM_008430) and mouse TASK-3 (NM_001033876) were obtained by using the Gateway cloning method (Invitrogen, Carlsbad, CA, USA). cDNAs encoding full-length human NTSR-1 (NM_002531) and human NTSR-2 (NM_012344) were synthesized by designing gene blocks (gBlocks^® Gene Fragments; Integrated DNA Technologies, Coralville, IA, USA) and constructing entry clones using the Gateway BP cloning method (Invitrogen). The constructs were cloned into several vectors, including pDEST-HA-N, pDEST-FLAG-N, pDEST-IRES2-GFP, and pDEST-IRES2-mCherry by using Gateway LR cloning (Invitrogen). To construct the concatenated TWIK-1 and TASK-3, TASK-3 and TWIK-1 were recloned into the pDONR207 P1P5R and pDONR207 P5P2 vectors, respectively, via two independent BP reactions (Invitrogen), followed by a MultiSite Gateway LR recombination reaction (Invitrogen) according to the manufacturer’s guidelines to produce pDEST-IRES2-GFP. The target regions of the shRNA (mouse TWIK-1: 5′-GCATCATCTACTCTGTCATCG-3′; mouse TASK-3: 5′-GCTGGTGTCCAGTGGAAATTC-3′) were obtained by oligonucleotide-directed mutagenesis using an EZchange site-directed mutagenesis kit (Enzynomics, Daejeon, Korea).

The 1.1‐kb promoter of PRT1 was amplified from genomic DNA with the primer pair AttB4_PRT1pro_For and AttB1R_PRT1pro_Rev and the full‐length genomic sequence of PRT1 without stop codon was amplified with primer pairs AttB1_PRT1_For and AttB2_PRT1_nonstop. These products were then recombined into pDONR P4‐P1R and pDONR221 vectors (Invitrogen) to generate pEN‐L4‐promPRT1‐R1 and pEN‐L1‐gPRT1‐L2, respectively. Both of the entry vectors were sequenced, and recombination reactions were carried out with pEN‐R2‐GStag‐L3 and pKCTAP (Van Leene et al., 2008) to generate construct MO14, using a Multisite Gateway^® LR Recombination Reaction (Invitrogen), according to manufacturer's instructions. Transformation into Agrobacterium tumefaciens (strain AGL‐1) and Arabidopsis thaliana prt1‐1 were performed according to established protocols (Clough & Bent, 1998), and transgenic plants were selected using kanamycin and subsequently methotrexate.