Pcr8 gw topo ta cloning vector

To generate the overexpression construct, a full-length cDNA of OsERF101 was amplified using the primers listed in Supplementary Table S1. The amplified fragments were ligated into the pCR8/GW/TOPO TA cloning vector (Invitrogen) and then inserted into the pMDC32 Gateway-compatible binary vector through the LR recombination reaction. The pMDC32-OsERF101 plasmids were introduced into callus of Dongjin seeds by Agrobacterium tumefaciens (strain LBA4404)-mediated transformation (Jeon et al., 2000 (link)). Agrobacterium-infected calli were transferred to 1/2 MS solid medium containing cytokinin and auxin. Plantlets regenerated from the callus were grown under continuous light conditions (90 µmole·m⁻² s⁻¹) at 30°C.

Full length SDA1 cDNA was amplified using Platinum Taq High Fidelity DNA Polymerase (Invitrogen, United States), with forward and reverse primers as listed in Supplementary Table S1, using U11998 SSP pUNI clone (ABRC) as template, and was cloned into pCR8/GW/TOPO TA cloning vector (Invitrogen, United States). Sequence of the amplified product was compared with the mRNA sequences available in the public databases to confirm that correct full-length cDNA was amplified. SDA1 cDNA was cloned in the gateway system pMDC32 vector (ABRC) (Curtis and Grossniklaus, 2003 (link)) under control of a strong 35S promoter to engineer SDA1 overexpression mutants. β-estradiol dose-dependent inducible overexpression of SDA1 was engineered by cloning of SDA1 cDNA in the gateway vector pMDC7 (Curtis and Grossniklaus, 2003 (link)). All of these constructs were then introduced into wildtype Col-0 plants via Agrobacterium-mediated floral dip transformation method (Clough and Bent, 1998 (link)). Transgenic plants were selected on solid Peters media or half-strength MS media with 1% sucrose and agar supplemented with 40 μg/mL hygromycin.
β-estradiol (Sigma, United States) treatment at indicated concentrations in 0.1% ethanol for inducible overexpression of SDA1, were syringe infiltrated into the rosette leaves of 4-week-old plants and tissue samples were harvested for RNA isolation at indicated times.

The SDA1 promoter was isolated as a 2 kb 5’-region upstream of the translation start site of SDA1 by PCR amplification of the genomic DNA using forward and reverse primers as listed in Supplementary Table S1. The amplified product was cloned into pCR8/GW/TOPO TA cloning vector (Invitrogen, United States), and sequenced to confirm the sequence of the amplified product. The promoter was cloned upstream of the GUS gene in the gateway system pMDC163 vector (ABRC) to create a transcriptional fusion SDA1 promoter::GUS. This construct was introduced into Col-0 plants via Agrobacterium-mediated transformation (Clough and Bent, 1998 (link)). Transgenic plants were selected on solid Peters media supplemented with 40 μg/mL hygromycin. Histochemical analyses for GUS activity were carried out in 6–8 independent transgenic lines for each tissue. Tissue samples were incubated overnight at 37°C in GUS assay buffer (10 mM phosphate buffer, pH 7; 0.5% Triton X-100; 2 mM potassium ferricyanide; 1 mg/mL X-Gluc) and were cleared in 70% ethanol for 2–3 days.

Genomic coordinates for deletion or fusion constructs of CNE17 are listed in the electronic supplementary material, table S2. GFP reporter constructs were generated as described in Pauls et al. [8 (link)]. Briefly, sequences were PCR-amplified from genomic DNA and cloned into the pCR8/GW/TOPO TA cloning vector (Invitrogen). This served as an entry clone for inserting the PCR products into a GFP expression vector described in Fisher et al. [22 (link)] allowing for tol2-mediated transgenesis in zebrafish [23 (link)]. In cases where stable transgenic zebrafish lines were analysed, each expression domain was confirmed in at least three independent lines. The RFP standard used for quantification of GFP lens expression was generated by cloning a lens enhancer linked to zebrafish sox2 (chromosome 22 : 40307301–40307520) and first identified in chicken [24 (link)]. The expression vector for the standard was generated by substituting the GFP sequence in the tol2 expression vector from [22 (link)] with an RFP sequence (gift from Javier Terriente). Mutagenesis was conducted following the QuickChange protocol using PfuUltra (Agilent Technologies) and the tol2 WT CNE clones as templates.

The CTG134 coding sequence (524 bp) was amplified by PCR from P. persica (cv. Red Haven, S4I development stage) cDNA and subsequently cloned into the pCR8/GW/TOPO TA Cloning vector (Invitrogen, Carlsbad, CA, United States). The CTG134 CDS was further inserted into a pGreen-derived vector (Hellens et al., 2000 (link)) with the Gateway cloning system (LR Clonase II – Invitrogen, Carlsbad, CA, United States). The pGreen-derived vector was modified to confer resistance to both kanamycin and ampicillin. Moreover, a CC_rfA gateway cassette was inserted downstream of the 35S promoter in the EcoRV site. As before, the selection of plants was carried out with kanamycin (Supplementary Figure 1B).