2.4 | Yeast-two-hybrid Experiment
The cDNA sequences of the large (StLS) and small (StSS) subunits of potato tuber AGPase44 and the large (OsL2) and small (OsS2b) subunits of rice endosperm AGPase45 were amplified through PCR using gene specific primers (Table. S3) and cloned into yeast-two-hybrid vectors, specifically pGBKT7 and pGADT7 (Figure S5). Following the preparation of cDNAs from immature rice seeds, the rice AGPase L1 (OsL1) and S1 (OsS1) subunits were amplified through PCR and subsequently cloned into the plasmid vectors. To perform protein-protein interaction experiments, a pair of plasmids carrying bait and prey sequences were co-transformed into Saccharomyces cerevisiae AH109 cells, according to the method described in.46 Subsequently, the cells, which were grown on SD medium lacking leucine and tryptophane (SD/-Leu-Trp), were transferred to SD/-Leu-Trp-Ade-His media for testing the protein-protein interaction.