Pmd18 t vector

Partial sequences of MrANR and MrLAR were isolated using degenerate primers (Table S1) designed from conserved regions of corresponding genes reported from other species by the CODEHOP strategy (Rose et al., 1998 (link)). The 5′ and 3′ ends of the coding sequence were obtained by rapid amplification of cDNA ends (RACE) using the SMART RACE cDNA amplification kit (Clontech, Mountain View, CA, USA). PCR fragments were subsequently cloned into pMD18-T vector (Takara, Japan) and sequenced. The complete open reading frames (ORFs) of MrANR and MrLAR were amplified from bayberry (Biqi) cDNA derived from fruit sampled at 113 DAFB PCR products were then ligated into the pMD18-T vector (Takara, Japan) and subjected to DNA sequencing. The primers used are shown in Table S1.

For fsGUS editing detection, genomic DNA was extracted from infiltrated leaves of KQ334 plants using Plant DNAsecure Plant Kit (TIANGEN Ltd, Beijing), and resuspended in 100 μL TE buffer (10 mM Tris/1 mM EDTA, pH 8.0). 300 ng genomic DNA was digested with ApaLI in a 50 μl volume, which were then purified by Multifunctional DNA Purification Kit (BioMED, Beijing). GUS DNA fragments were PCR amplified using purified DNA as template with primers oYK700 and oYK701, and cloned into pMD18-T vector (Takara), followed by DNA sequencing to check mutations. For detection of NbPDS editing, genomic DNA extracted from systemic leaves was used as PCR template to amplify NbPDS locus with primers PDS_MlyIF and PDS_MlyIR10 (link) and EasyTaq DNA polymerase (Transgene, Beijing). PCR product was purified and digested with MlyI and run on a 2% agarose gel. Mutation rate was estimated by ImageJ (NCBI). The uncut DNA band was gel purified by DNA purification kit (BioMed, Beijing), subsequently cloned into pMD18-T vector (Takara). DNA sequencing was used to check mutations.

The ORFs of GlPGM1 and GlPGM3 were amplified, as mentioned above, and then cloned into the pMD18-T vector (Takara, Dalian, China) for sequencing (Youkang Biotech, Hangzhou, China). The GlPGM1 and GlPGM3 from the pMD18-T vector were then cut, using restriction endonuclease and cloned into expression vector pET-28a (+) (Takara, Dalian, China) to obtain pET-28a-GlPGM1 and pET-28a-GlPGM3 recombinant strains, respectively. The constructed plasmids were transformed into the expression strain E. coli BL21 (Takara, Dalian, China). The recombinant strains of GlPGM1and GlPGM3 were incubated and induced with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3–5 h at 28 °C. The overexpressed proteins GlPGM1and GlPGM3 were purified and checked by 12.5% SDS-PAGE. The E. coli with recombinant pET-28a without IPTG was used as the negative control.
The GlPGM1 and GlPGM3 polyclonal antibodies were made by Hangzhou Aiting Biological Technology Co., Ltd. (Hangzhou, China). The purified proteins, GlPGM1 and GlPGM3, were used as antigens to immunize rabbits for the production of the polyclonal antiserum. A Western blot analysis with the antibodies was conducted to check the expression of GlPGM1 and GlPGM3 from G. lemaneiformis (Figure S7, Supplementary Materials).

Approximately 5 μg of the ApoE Cas9-sgRNA targeting plasmid was co-transfected with 1 μg of the neomycin expression plasmid (pCMV-tdTomato) into 1×10⁶ early passage of PFFs using the Basic Fibroblast Nucleofection Kit (Amaxa Biosystems/Lonza, Cologne, Germany), according to the manufacturer's instructions. A total of 800 μg/ml of G418 (Gibco, Grand Island, NY, USA) was applied to the post-transfection cells 48 h later and maintained for 10-14 days thereafter. The G418-resistant colonies were seeded in 24-well plates and then passaged to 12-well plates. Approximately 1/5 of the cells of a single colony were lysed with NP-40 (55°C for 30 min, 95°C for 10 min) for PCR screening and 4/5 of the remaining cells were used for SCNT. The primers used in amplifying the target regions were as follows: forward: 5′-CCTCAGGTGGTCTAGGTTGG-3′, reverse: 5′-TTTTGCAATGGAGGAGTGGC-3′. The PCR conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 50 s, and a final 72°C for 4 min. The PCR products were subcloned into a pMD18-T vector (Takara Clontech, Tokyo, Japan) according to the manufacturer's instructions. Fifteen to 20 individual clones were picked and sequenced.