Pce2 ta blunt zero vector

Specific primers for BoNA1 (BoNA1-F/R) were designed by Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/) and were used to amplify the full-length gene sequences from YL1, BK2019, the transgenic lines and the other kale varieties. PCR amplification and PCR protocol were performed through the methods described by Yi [28 ]. The PCR products were then cloned into a pCE2 TA/Blunt-Zero Vector (Vazyme, Nanjing, China). After transformation into FastT1 competent cells, approximately 60 white colonies were randomly picked, and plasmids were extracted from the positive clones by using TIANprep Rapid N96 (TIANGEN Biotech, China). The plasmids were sequenced via the Sanger method by the Beijing Genomics Institute (Beijing, China). Sequence alignment was subsequently performed with DNAMAN version 6.0 software and Chromas software v2.31 (https://www.technelysium.com.au/chromas.html).
The full-length amino acid sequence of BoNA1 was used to search for relatives based on NCBI-based BLASTP searches. Phylogenetic trees were constructed using MEGA7.0 software [45 (link)] with the Neighbor-Joining and 1000 replicated bootstrap.

To identify the S-RNase allele in N. alata, degenerate primers C2F/C4R (Supplementary Table 1) were used to amplify the S-RNase candidates. Genomic DNA was extracted from N. alata leaf tissue using the Plant Genomic DNA Miniprep Kit protocol (Tiangen, Tianjin, China). Genomic PCR was performed using the Q5 High-Fidelity DNA Polymerase, as described above. The amplified PCR product was cloned into the pCE2-TA-Blunt-Zero vector (Vazyme, Nanjing, China) following the manufacturer’s instructions. 10 Clones of each plant were sequenced using Sanger sequencing platform (Generay, Shanghai, China), and the obtained sequences were subjected to BLAST search against the non-redundant NCBI database to determine the candidate S-RNase allele.
To confirm the S2/Sc10 bi-allele distribution result in the family, specific primer pairs S2F/R and Sc10F/R (Supplementary Table 1) were used to examine the same population. The PCR products displaying expected size unique bands were subjected to electrophoresis using the ZAG DNA Analyzer (Agilent, Santa Clara, USA).

The genomic DNA of the stable transgenic N. alata plants from hygromycin selection and wild-type plants were extracted from leaf tissue using the Plant Genomic DNA Miniprep Kit protocol (Tiangen) to assess targeted mutagenesis using PCR amplification and Sanger sequencing. The genomic region spanning the CRISPR target sequences were amplified by PCR (primer sequences in Supplementary Table 1) using Q5 High-Fidelity DNA Polymerase (NEB). The PCR products were sequenced and then subjected to SnapGene software assay (www.snapgene.com) according to the manufacturer’s instructions. PCR fragments of putative editing events were then cloned into pCE2-TA-Blunt-Zero vector (Vazyme) and 10 clones each plant were sequenced to further measure the frequencies of CRISPR induced mutations. The mutation rate was calculated based on the ratio of mutated plants versus total transgenic plants.

DNA was extracted from gut homogenates using CTAB method (58 (link)). DNA concentration was determined with Qubit 4 Fluorometer (Thermo Fischer Scientific; Waltham, MA, USA). H. alvei loads were determined by qPCR using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China). H. alvei-specific primer sets are listed in Table S1 in the supplemental material. All qPCRs were performed in 96-well microplates on a QuantStudio 1 real-time PCR system (Thermo Fischer Scientific). Melting curves were generated after each run (95°C for 15 s, 60°C for 20 s, and increments of 0.3°C until reaching 95°C for 15 s). Standards for H. alvei 16S rRNA and the host’s actin were prepared by cloning sequences into the pCE2 TA/Blunt-Zero Vector (Vazyme Biotech). Each reaction was performed in triplicates on the same plate. The data was analyzed using the QuantStudio Design and Analysis Software. After calculating H. alvei 16S rRNA gene copies, normalization was performed to reduce the effect of gut size variation and extraction efficiency using the host’s actin gene (59 (link)).

The amino acid sequences of SMV strains G7, G7d, and N were retrieved from Genbank under the accession nos. AY216010, AY216987, and D00507, respectively. The HcPro and P3 of SMV N3 were amplified from total RNA extracted from leaves of Dongnong 50 that infected by SMV N3 using the Phanta max super-fidelity DNA polymerase (Vazyme) with the primers shown in Table 1. The amplified fragments were ligated into pCE2-TA-Blunt-Zero vector (Vazyme) and Sanger sequenced at RuiBiotech Co., Ltd. (Beijing, China). Amino acid sequences were aligned with Clustal Omega with default settings [31 (link)].

Based on the annotated coding sequence (CDS) in the genome database (Yang et al., 2021 (link)), we cloned the CDS of CsSEP1, CsSEP2, CsSEP3, and CsSEP4 using specific primers designed by PrimerPremier 5.0. Total RNA was extracted from natural flowers of C. sinense ‘Baimo’ using the RNAprep Pure Plant Kit (TIANGEN, China) following the manufacturer’s instructions. RNA content was measured with a Nano-Drop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), and 1 μg of RNA was used to synthesize first-strand complementary DNA (cDNA) with the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China). The cDNA was then used as a template to clone the CDS of CsSEP genes with high-fidelity Taq DNA polymerase (Vazyme, China), and the gene-specific primers used are listed in Supplementary Table S3. The PCR products were purified using the FastPure^® Gel DNA Extraction Mini Kit (Vazyme, China) and cloned into the pCE2 TA/Blunt-Zero Vector (Vazyme, China) for transformation into DH5α (Tiangen, China). Positive clones (8-10) were selected for identification and sequenced by the Sangon Company in Shanghai. The plasmids were extracted with the FastPure^® Plasmid Mini Kit (Vazyme, China).