One step cloning kit

The coding sequence of PpSnRK1βγ without stop codon was amplified from ‘JH’ using PCR primers listed in Table S2 and in-frame fused to the N-terminal of the GFP in the plant binary expression vector pBWA(V)HS-Glosgfp to generate the 35S:PpSnRK1βγ-GFP construct. One step cloning kit (Novoprotein, China) was used to form recombination vector. The 35S:GFP construct in the original vector was used as a control and 35S:NLS-mkate was used as a nucleus localization marker. Agrobacterium tumefaciens strain GV3101 was transformed with these three vectors separately and cultivated in LB medium containing 50 μg/mL kanamycin under 28 °C. After cultivation, the Agrobacterium cells were re-suspended with infiltration buffer containing 10 mM MgCl₂, 10 mM MES, and 200 μM acetosyringone to OD600 of 0.6–1.0, and placed at room temperature for 2 h. The cells containing 35S:PpSnRK1βγ-GFP and 35S:GFP were mixed with the same volume of cells containing 35S:NLS-mkate, and injected into leaf tissues of tobacco (N. benthamiana) using a 1-mL syringe without needle. After infiltration, the tobacco plants were firstly placed in dark at room temperature for 12 h and then moved to conditions of 16 h light and 8 h dark for 48 h. The GFP and mkate (monomeric version of Katushka) fluorescence were observed using a confocal laser scanning microscope (TCS SP5, Leica, Germany).

For complementation plasmid construction, the full-length CDS of AtSCC2 or two separated fragment CDS of AtSCC2 lacking PHD domain (AtSCC2ΔPHD) was cloned into modified plasmid pCAMBIA1306 (AtACT7::3*FLAG) by One-step Cloning Kit (Novoprotein, China). To generate transgenic AtSCC4-RNAi plants, two regions of AtSCC4 CDS were amplified using PCR with the primers including NcoI/XbaI and SpeI/SalI restriction sites, respectively. The amplification products were ligated into the pMeioDMC1-intron vector using SpeI/NcoI for the sense fragment and SalI/XbaI for antisense fragment. The constructs were then individually transformed into Agrobacterium tumefaciens GV3101 (Weidi, China) and bacterial cultures were used for dip transformation as previously reported [82 (link)]. Positive T1 plants were screened on 1/2 MS medium containing 25 mg/L hygromycin.