The ORF (open reading frame) of TaWRKY13 was amplified by PCR with specific primers from wheat cDNA (cultivar Jinhe 9123). The PCR product was fused into pZeroBack vector (TIANGEN, Beijing, China) and sequenced for further study. The correct sequencing plasmids were treated as templates, the segment with restriction sites was amplified by specific primers, and the PCR product was inserted into the N-terminus of the green fluorescent protein (GFP) containing the CaMV35S promoter for subcellular localization; the 35S::GFP vector was used as the control. Both 35S::GFP and 35S::TaWRKY13-GFP were transferred into wheat mesophyll protoplasts by the PEG-mediated method [29 (link)]. A confocal laser scanning microscope (LSM700; CarlZeiss, Oberkochen, Germany) was used to observe the fluorescence after incubation in darkness at 22 °C for 18–20 h. All primers are listed in Supplementary Table S4 .
Pzeroback vector
Manufactured by Tiangen Biotech
Sourced in China
The PZeroBack vector is a plasmid designed for gene cloning and expression. It serves as a backbone for inserting and maintaining DNA sequences in bacterial hosts. The vector contains key elements such as an origin of replication and antibiotic resistance markers to facilitate selection and propagation of the recombinant construct.
Automatically generated - may contain errors
2 protocols using pzeroback vector
1
Subcellular Localization of TaWRKY13 in Wheat
2
Full Genome Sequencing of SFTSV and NSDV
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To obtain the full genomes of SFTSV and NSDV, previously developed primer pairs were used in the PCR systems with the Fast HiFidelity PCR kit (Tiangen) (Gong et al., 2015 (link), Liu et al., 2016 (link)). The amplicons were blunt ligated into the pZeroBack vector (Tiangen), and used to transfect competent E. coli DH5α (Tiangen). Three clones of each amplicon were randomly picked for sequencing. Overlapping amplicons were assembled with SeqMan v7.0 (DNAstar).
Genomic structure predictions and phylogenetic characterizations were conducted using our previously published methods (Yang et al., 2018 (link)). Briefly, genomic structures were predicted by Vector NTI v.11.5, and then validated by comparison with genomes of other SFTSV and NSDV. Nucleotide reference sequences were retrieved from Genbank and aligned with counterparts of viruses identified here using MAFFT v7.394 (Katoh and Standley, 2013 (link)). Evolutionary models were determined using ModelGenerator v0.85 with Akaike Information Criterion 1 (AIC1) (Keane et al., 2006 (link)). Phylogenetic analyses were conducted using PhyML v3.3 by the maximum likelihood and best-fit substitution models with evaluation of 100 bootstraps, and visualized with FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree ). Nucleotide and amino acids identities were calculated using MegAlign v7.1 (DNASTAR).
Genomic structure predictions and phylogenetic characterizations were conducted using our previously published methods (Yang et al., 2018 (link)). Briefly, genomic structures were predicted by Vector NTI v.11.5, and then validated by comparison with genomes of other SFTSV and NSDV. Nucleotide reference sequences were retrieved from Genbank and aligned with counterparts of viruses identified here using MAFFT v7.394 (Katoh and Standley, 2013 (link)). Evolutionary models were determined using ModelGenerator v0.85 with Akaike Information Criterion 1 (AIC1) (Keane et al., 2006 (link)). Phylogenetic analyses were conducted using PhyML v3.3 by the maximum likelihood and best-fit substitution models with evaluation of 100 bootstraps, and visualized with FigTree v1.4.3 (
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