Pcr 8 gw topo ta vector

The purified PCR products of SGT5019, SGT9704, and Pk1b amplified from maize B73 cDNA were cloned into pCR8/GW/TOPO TA vector following the manufacturer’s protocol (Thermo Fisher Scientific). The resulting ligation mixtures were transformed into E. coli and positive clones were identified by colony PCR. Positive clones were then subcloned into pGWB641 featuring a recombinant C terminal EYFP tag (Nakamura et al., 2010) using LR clonase II (Thermo Fisher Scientific). Positive clones were then transformed into Agrobacterium tumefaciens GV3101.

The full-length Mepul was cloned into a pCR™8/GW/TOPO^® TA vector (Invitrogen™) based on circular polymerase extension cloning technique (Aslanidis and De Jong, 1990 (link); Quan and Tian, 2011 (link)). The gene with LR sequences was then subcloned into a pGWB5 vector fused with the C-terminal green fluorescent protein (GFP) (Nakagawa et al., 2007 (link)) using Gateway^® LR Clonase^® II (Invitrogen™). The pGWB5-Mepul was transformed into an Agrobacterium tumefaciens GV3101 by electroporation.
A. tumefaciens harbouring pGWB5-Mepul and A. tumefaciens containing the silencing suppressor p19 gene were co-infiltrated into 4-week Nicotiana benthamiana leaves (Lindbo, 2007 (link); Panpetch and Sirikantaramas, 2021 (link)). A pGWB2 bearing GFP gene was used as a control for infiltration experiments. After 3 days, localisation of MePUL was observed under a FluoView^® FV10i-DOC confocal laser scanning microscope (Olympus Corp.). GFP fluorescence signal was monitored at 488/510 nm of excitation/emission, while chloroplast autofluorescence was observed at 633/664 nm.

DNA fragments containing full-length coding sequences of MtCOLa-COLh were amplified by PCR from cDNA and cloned into the pCR8/GW/TOPO TA vector (Invitrogen). The resulting entry vector was then recombined into plant transformation vector, pB2GW7 (Karimi et al., 2002 (link)) to generate the 35S:MtCOLa-h constructs. Transgenic plants were produced by applying Agrobacterium tumefaciens strain LBA4404 containing the pB2GW7 vectors to Arabidopsis co-2 mutant flowers using the protocol described by Martinez-Trujillo et al. (2004 (link)). Seeds from these plants were collected and sown directly onto soil and selected using Basta herbicide. Putative transformants were confirmed by qRT-PCR analysis.

To generate the 35S::XAL1 (without a tag), the full-length XAL1 cDNA was amplified using the 5′-ATTGGATCCATGGCTCGTGGAAAGATTCAGC-3′ and 5′-TTAGGATCC CTAGAACTGAAATATTTCACTTG-3′ primers that contain the BamH1 restriction sites. DNA was cloned into the p-GEM-T Easy vector (Promega, Madison, WI, USA) and, after excision, it was subcloned into the pBIN-JIT vector carrying a double 35S promoter. The correct clone orientation was confirmed by sequencing [79 (link)]. For the 35S::XAL1::GFP construct, the XAL1 cDNA (without the stop codon) was obtained using the XAL1-F 5′-ATGGCTCGTGGAAAGATTCAGC-3′ and the XAL1-R 5′-GAACTGAAATATTTCACTTGGCA-3′ primers. The resulting 633 bp DNA fragment was cloned into the pCR8/GW/TOPO-TA vector (Invitrogen, Thermo Fisher Scientific) and verified via sequencing. Subsequently, it was recombined into the overexpression vector pGWB5 which carries the 35S promotor and the GFP gene using the Gateway LR system (Invitrogen, Thermo Fisher; [80 (link)]). Both constructs were introduced into Agrobacterium tumefaciens (C58) and then transformed into the xal1-2 plants using the floral-dip method [81 (link)]. Kanamycin resistant plants were selected on MS 0.2x plates (MP biomedicals, Santa Ana, CA, USA) and T2 transgenic lines were chosen based on the complementation of the xal1-2 short root phenotype grown on sulfadiazine (20 mM) plates.

Standards of delphindin 3-rutinoside (Extrasynthese, Genay, France), chlorogenic acid, nicotine, and tryptophan (Sigma, St Louis, MO, USA) were used in concentrations ranging from 3.12 to 200 μg/ml. cDNAs from ROS1 and DEL were amplified by PCR using genomic DNA of E8:ROS/DEL tomato fruits obtained from (Butelli et al., 2008 (link)). PCR products were gel purified and TOPO cloned into the pCR8/GW/TOPO-TA vector (Invitrogen). After sequence verification the ROS1 and DEL fragments were transferred by GATEWAY recombination to pK7WG2 ¹ to create 35S-ROS1 and 35S-DEL. The plasmids obtained were then introduced into Agrobacterium tumefaciens AGL0 (Lazo et al., 1991 (link)). AGLO harboring pBINPLUS (pBIN) plasmid (van Engelen et al., 1995 (link)) was used as a negative control.