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.