4.3. Yeast Two-Hybrid Assays

Yeast two-hybrid assays were performed using yeast AH109 nutrition deficient strain. The OsMTA2 and OseIF3h coding sequences were amplified, respectively. PCR products were then cloned into pGBKT7 and pGADT7 (Clontech) vectors to form bait and prey constructs using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). All constructs were confirmed by sequencing. Empty vectors were used as negative controls. All bait and prey construct pairs were transformed into the yeast AH109 strain via PEG/LiCl mediated transformation methods. The yeast cells were cultured at 28 °C on SD/-Trp-Leu for successful transformation testing before they are dotted onto SD/Trp-Leu-His-Ade medium plates for interaction evaluation.