HEK293T, NIH3T3, RAW264.7 and A549 cells were obtained from American Tissue Culture Collection (ATCC). Irf3−/−Irf7−/− MEFs were kindly provided by Dr. Pinghui Feng (University of Southern California). Sendai virus (SeV) and vesicular stomatitis virus (VSV) were kindly provided by Dr. Hong-Bing Shu (Wuhan University). Herpes simplex virus-1 (HSV-1) were kindly provided by Dr. Yu Liu (Wuhan University). Murine hepatitis virus (MHV) strain A59, which is positive single-stranded RNA (+ssRNA) virus and belongs to the beta coronavirus in Nidovirales that includes Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), was used as described previously [44 (link), 45 (link)]. The chemical reagents used in this study were from the following manufacturers: poly(I:C) (Invivogen); ssPolyU (Invivogen); One Step Seamless Cloning kit (Aidlab,China); GM-CSF (Peprotech); TRIzol (Invitrogen).
One step seamless cloning kit
Manufactured by Aidlab
Sourced in China
The One Step Seamless Cloning kit is a laboratory tool designed for the rapid and efficient cloning of DNA fragments. It provides a streamlined process for the ligation and transformation of DNA into competent cells, enabling the creation of recombinant plasmids.
Automatically generated - may contain errors
Lab products found in correlation
Gm csf, by Thermo Fisher Scientific (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Sspolyu, by InvivoGen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Nih3t3, by American Type Culture Collection (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Lsm510 meta confocal laser microscope, by Zeiss (1 mentions)
Sp8 microscope, by Leica camera (1 mentions)
5 protocols using one step seamless cloning kit
1
Immune Response Activation Protocols
2
Interaction Detection Between MdERF3 and MdERF118
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The full‐length CDS of MdERF3 or MdERF118 without stop codon was amplified and ligated into pSPYNE‐35S and pSPYCE‐35S vectors containing green fluorescent protein (GFP), using the one‐step seamless cloning kit (Aidlab Biotechnologies); the constructs were checked by sequencing and then transformed into A. tumefaciens strain GV3101. Two plasmids were co‐transformed into the abaxial side of 4‐ to 6‐week‐old N. benthamiana leaves to detect specific interactions as described previously (Zhang et al., 2013 ). After 48 h co‐infiltration, the N. benthamiana leaves were observed using an LSM 510‐Meta confocal laser microscope (Zeiss). GFP signals were imaged under 488 nm excitation wavelength.
3
Transient expression of fluorescent fusion proteins
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ZjVND7 and ZjSMR1 were recombined and ligated into a pYBA1152 vector labeled with eGFP using a one-step seamless cloning kit (Aidlab, China). In short, primers with overlapping sequences were designed for PCR amplification of full-length CDS sequences. Then, the gel was recycled, and a reaction (10 μl volume) was performed to ultimately connect it to the carrier according to the instructions. The correctly sequenced plasmid was transformed into Agrobacterium. Then, an MES suspension (OD = 0.5–1.0) was injected into 1-month-old tobacco leaves. The plants were incubated in the dark for 24 h under a 16/8 h light/dark cycle for 48h at 25 ± 2°C. The injection sites were then removed and observed under a SP8 microscope (Leica, Wetzlar, Germany).
4
Overexpression of MdSAUR36 in Apple
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Full length CDS of MdSAUR36 was enzyme digested from cloning vector by HindIII/SalI or HindIII/BamHI, and then were linked to the multiple cloning site (MCS) of PRI-101-GFP or PRI-101-RNAi using One Step Seamless Cloning kit (Aidlab Biotechnologies, Beijing, China). Transgenic apple calli (derived from mesocarp of cultivar 'Orin') were obtained by Agrobacterium-mediated transformation method. Plant expression vectors and empty vectors were transformed into Agrobacterium tumefaciens strain GV3101 by heat shock. The apple calli were infected and co-cultured with the transformed Agrobacterium tumefaciens at 25°C in dark for two days, then cultured on the screening medium with kanamycin and cephalosporin resistance for a least 20 days until positive calli appear to grow. The positive calli were sub-cultured on the screening medium for 3-4 times. The successful transgenesis was con rmed by RT-qPCR.
5
Genetic Constructs for Promoter Activity Analysis
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To determine the impact of the allelic variations in the upstream regions of MdERF3 and MdERF118 on promoter activity, genetic constructs with the promoters of MdERF3 and MdERF118, and also their CDS, respectively, plus a GUS reporter, were prepared using the one‐step seamless cloning kit (Aidlab Biotechnologies, Beijing, China). These included MdERF3pro‐F:GUS (Del323), MdERF3pro‐F:GUS (del323), MdERF118pro‐Z:GUS (Del229), and MdERF118pro‐Z:GUS (del229). All constructs were transiently transformed into apple calli (from hypanthium of ‘Orin’ cultivar) using the method described by Jia et al. (2018 ). The complete MdERF3 and MdERF118 CDS cDNA sequences were also amplified and cloned into the NdeI/EcoRI and SalI/BamHI sites of the PRI101 vector, respectively. Partial MdERF3 and MdERF118 CDS sequences were cloned using the restriction enzyme sites XbaI/SalI, EcoRI/SalI, and XbaI/SalI, KpnI/EcoRI of the RNAi vector. The primer pairs used are listed in Table S16 .
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