2.7 Yeast one-hybrid assay (Y1H)
Yeast one-hybrid assay (Y1H) was performed to verify the DNA-protein
interaction following the manual of the Matchmaker one-hybrid system
(Clontech). The full-length cDNA of OsLHY was cloned to fuse with
the GAL4 activation domain in the pGAD7-Rec2 vector. The promoters of
flowering related genes were amplified to insert into the pHIS2 vector
to generate reporter constructs. Subsequently, these reporter plasmids
were separately co-transformed with the fusion plasmid pGADT7-OsLHY into
the yeast Y187. The co-transformed yeast cells were grown on the
SD/-Leu/-Trp/-His and SD/-Leu/-Trp/-His media containing 50 mM
3-amino-1,2,4-triazole (3-AT) in a 30℃incubator for 3-5 d. Relative
primers are shown in Supplemental
Table 1.