Quick easy yeast transformation mix kit

According to the instructions of ClonExpressII One Step Cloning Kits (Vazyme, Nanjing, China), the full length, N-terminal (1–526 bp) and C-terminal (527–1059 bp) of the RcNAC72 gene were inserted into the EcoRI and BamHI of the pGBKT7 vector. The recombinant plasmids and pGADT7 plasmid were transferred into Y2HGold yeast cells (Huayueyang, Beijing, China), referring to the Quick Easy Yeast Transformation Mix kit instructions (Clontech, San Jose, CA, USA). The transformed yeast cells were diluted 10-fold with sterile water and 10 μL of the diluted solution was spotted on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade-x-α-gal media, respectively. These were cultured upside down at 30 °C for 3 days, and the yeast growth was observed.
The full length of RcDREB2A was inserted into pGBKT7 vector as a prey, and the full length of RcNAC72 was inserted into pGADT7 as a bait. As mentioned above, pGBKT7- RcDREB2A and pGADT7- RcNAC72 plasmids were jointly transferred into Y2H yeast. These yeast cells were observed on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade-x-α-gal selective media. The primers used above are listed in Table S1.

The ORF of LoSWEET14 without a stop codon was inserted between the EcoRI and SpeI restriction sites of the pDR196 vector. The pDR196-LoSWEET14 fusion plasmid and the empty vector were transferred into the EBY.VW4000 yeast strain referring to the Quick Easy Yeast Transformation Mix Kit (Clontech, Mountain View, CA, USA), respectively. Transformants were screened in an SD/-Ura plate added with 2% maltose and the transformed cells were cultured in an SD/-Ura liquid medium to OD = 0.5, then it was diluted and spot coated in SD/-Ura plates containing 2% maltose, 2% sucrose, 2% glucose, and 2% fructose, respectively. The growth of yeast cells was observed after three days incubation at 30 °C.