Kanamycin monosulfate

A 2.369 kb fragment containing a 5ʹ VEGFA sequence from −2304 to +65 relative to the transcription initiation site was amplified by PCR using Q5 High-Fidelity DNA Polymerase (NEB, United States). The forward primer with a SalI site was 5ʹ-GATGTCGACTTGCTGGGTACCACCATGGA-3ʹ, and the reverse primer, which had a XbaI site, was 5ʹ-GATTCTAGACAGAGCGCTGGTGCTAGCC-3ʹ. After digestion by QuickCut restriction endonucleases (Takara, United States), a DNA Ligation Kit (Takara, United States) was used to insert the PCR sequences into the SalI and XbaI sites of pEZX-FR01 (GeneCopoeia, United States), which contains a Rinella luciferase (Rluc) coding sequence with a CMV promoter and a promoter-less firefly luciferase (HLuc) coding sequence.
These recombinant plasmids were transfected into DH5α cells (TSINGKE, China) and amplified in nutrition agar plate (Solarbio, China) culture with kanamycin monosulfate (50 μg·mL⁻¹, Solarbio, China) to select different monoclonal bacterial colonies. Then, the selected single clones were cultured in LB medium (Solarbio, China) with kanamycin. The recombinant plasmids were purified by a plasmid extraction kit (TIANGEN, China). All plasmid constructs were verified by direct sequencing (Sangon Biotech, Chengdu). The reporter plasmid was designated pEZX-VEGFA.

Restriction enzymes Sac I, SnaB I and Not I were purchased from Takara Bio Inc., Japan, and used as detailed by the manufacturer. Yeast nitrogen base w/o amino acids (YNB), D-sorbitol, Kanamycin monosulfate, Zeocin and geneticin sulfate (G-418S) were obtained from Solarbio, Beijing, China. Oxoid™ peptone and yeast extract were purchased from Thermo Scientific, Germany.
P. pastoris was grown in yeast peptone dextrose medium (YPD, 1% yeast extract, 2% peptone and 2% dextrose) or buffered complex glycerol medium (BMGY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 0.4 mg/mL biotin, 1% glycerol). P. pastoris was induced in buffered complex methanol medium (BMMY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 0.4 mg/mL biotin, 0.5% methanol). YPD plates containing 1 M Sorbitol and 2.5 mg/mL geneticin sulfate were used for selection of positive strains containing the pPIC3.5 K expression vector. E. coli was cultivated in LB medium (0.5% yeast extract, 1% peptone, 1% NaCl). F. graminearum was also grown in YPD medium.