Clone express

Human codon-optimized DNA sequences encoding M13, f1, f1 mutant 2, and pf1 G8P_PD were cloned into the BamHI/XbarI sites of pcDNA3.1(+) by plasmid recombination kit Clone Express (Vazyme). These G8P_PD peptides carry an N-terminal SV40 nuclear localization signal (NLS) for co-localization with Cas9 proteins. G8P_PD peptides were cloned into plv-EF1α-mCherry plasmid harboring mCherry fluorescent protein marker. sgRNA was cloned into pGL3-U6-gRNA plasmid carrying green fluorescent protein (GFP).

sgRNA was cloned into pGL3-sgRNA expression vector carrying a U6 promoter and an EGFP reporter gene (Addgene, #107721). CBE-targeted genomic sites are indicated in Supplementary Table 1. The sequences of sgRNA-encoding oligonucleotides were listed in Supplementary Table 2. Human codon-optimized DNA sequences encoding M13, f1 G8P_PD and Mut2 G8P_PD were cloned into the BamH I/Xba I sites of pcDNA3.1(+) by plasmid recombination kit Clone Express (Vazyme Biotech, Nanjing, China). These G8P_PD peptides carry a C-terminal SV40 nuclear localization signal (NLS) for co-localization with Cas9 proteins. G8P_PD peptides were cloned into plv-EF1α-mCherry plasmid harboring mCherry fluorescent protein marker. For construction of EGFP-Y66C reporter plasmid, Y66C mutation was introduced by quickchange PCR. Mut2 G8P carrying a C-terminal SV40 nuclear localization signal (NLS) was cloned into A3A CBE expression vector.

To further study the function of the MAPK gene of Tartary buckwheat, we selected MAPK1 and cloned the complete coding sequence. The open reading frame (ORF) of FtMAPK1 was PCR-amplified using primers (Additional file 4). Then, the sequence was inserted into the vector, pCHF3-YFP with Clone Express (Vazyme, C112-02). The recombinant plasmid was mediated by Agrobacterium tumefaciens to transform Tartary buckwheat leaves. The empty vector pCHF3-YFP was transiently transformed under the same conditions as those used for the negative control.
Three days after transformation, the leaves of the experimental group and the control group were placed on MS plates containing 200mM NaCl for 24h, and then the superoxide dismutase (SOD) and peroxidase (POD) content of the experimental group and the control group were determined. SOD and POD were determined by the previous methods [55 ].