T4 ligase

The plasmid pET28a–Vip3Aa, which carries Vip3Aa11 (NCBI accession No. AAR36859), was used as the template to generate the expression plasmids for Vip3Aa mutants. The Vip3Aa protein structure (PDB: 6TFJ) reported by Núñez–Ramírez et al. was used as a reference for selecting amino acid mutation sites [7 (link)]. Two kinds of mutant vectors (loop replacement mutations and point mutations) were constructed in this study. Take the β4–β5 loop of Domain III in Vip3Aa as an example to expound the plasmid construction of loop replacement mutations. In brief, the loop sequence substitution (mutation of residues 364–DSI–368 to 364–AAA–368) was obtained by PCR amplification using the primers loop 4–5A–F/loop 4–5A–R. T4 ligase (TransGen Biotech, Beijing, China) was then used to ligate the phosphorylated PCR products to obtain the β4–β5 loop replacement mutant plasmid pET28a–Vip3Aa–loop 4–5A. All point mutation vectors were obtained using the Fast MultiSite Mutagenesis System (TransGen Biotech, Beijing, China). All primers used for plasmid construction in this study are listed in Table S1.

FcCHS1, FcCHI1, FcDFR1, FcMYB21, and FcMYB123 were cloned based on the putative ORFs of fig unigenes from our RNA-seq data. The specific primer pairs (Table S8) were designed to amplify the ORF sequences using KOD DNA polymerase (TOYOBO, Osaka, Japan) from the cDNA of red fig peels. The reaction conditions were as follows: 96 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, with 10 min extension at 72 °C. The PCR products were cloned into the pMD19-T simple vectors (TaKaRa, Shiga, Japan). Afterwards, those T-vectors were transferred into DH5α competent cells (Transgen Biotech, Beijing, China) for amplification, and the products were sequenced by GENEWIZ (New Jersey, USA).
The overexpression vectors of FcMYB21 and FcMYB123 were constructed via linking their ORFs into the linearized plant transformation vector pBI121 using fast-digest restriction enzymes of XbaI and BamHI (Thermo Scientific, Waltham, USA) and T4 ligase (Transgen Biotech, Beijing, China). Then the 35S::FcMYB21 and 35S::FcMYB123 recombinant vectors were transformed into Agrobacterium tumefaciens EHA105 competent cells, respectively.

DMEM medium, fetal bovine serum, T4 ligase, T7 endonuclease, Escherichia coli DH5α, plasmid extraction kit, genomic DNA extraction kit, reverse transcription kit, and SYBR^® Green RT-PCR Master Mix were purchased from TransGen Biotech Co (Beijing). ACCUTASE™ cell digestion solution was purchased from eBioScience Inc. Western blot antibodies, STAT1 (14994), p-STAT1 (Tyr701, 7649P). STAT3 (9132), p-STAT3 (Tyr705, 9145), STAT5 (9363p), p-STAT5 (Y694, 4322). AURKA (14475), p-AURKA (Thr288, 3079), PD-L1 antibody (13684), p-AKT (Ser473, 4060), AKT (pan) (4691), p-ERK/ERK antibody (4370), GAPDH (5174), β-tubulin (2128) were purchased from Cell Signaling Technology, CD3 histochemical antibody (100219), CD8 histochemical antibody (104705) were purchased from Biolegend, and 4% paraformaldehyde were purchased from Wuhan Seville Biological LTD. DAB histochemical staining kit was purchased from Zhongshan Jinqiao Biological Technology LTD (Beijing). ECL luminescence color reagent was purchased from Healthcare. Trizol RNA extraction reagent was purchased from Sigma-Aldrich (T9424). Anti-mouse PD-L1 Antibody was purchased from BioXcell.