Study of Interaction between OsRUS1 and OsRUS2.1 by Yeast Two-Hybrid Technology

Yeast two-hybrid technology was applied to study whether OsRUS1 interacts with OsRUS2.1,and they interact through which domain.The four different length of OsRUS1 fragments [OsRUS1(1-1 782),OsRUS1(1-504),OsRUS1(510-1 282),OsRUS1(1 188-1 782)] were cloned into bait vector pGBKT7,and four different length of OsRUS2.1 fragments [OsRUS2.1(1-1 317),OsRUS2.1(1-138),OsRUS2.1(139-879),OsRUS2.1(880-1 317)] were cloned into prey vector pGADT7,according to the distribution of DUF647 domain in OsRUS1 and OsRUS2.1.These constructed vectors were confirmed by enzyme digestion and sequencing.The four OsRUS1 bait vectors and four OsRUS2.1 prey vectors were transformed into yeast strain AH109,respectively.The self-activation and toxicity of the plasmids to host yeast AH109 were analyzed by the expression of ADE,HIS,MEL1 and LacZ reporter genes and by culturing in auxotroph medium SD-Trp-DO or SDLeu-DO.Results showed that the eight constructed vectors had no self-transcriptional activity and not toxicity to yeast strain AH109.Then 16 combinations of four OsRUS1 bait vectors and four OsRUS2.1 prey vectors were cotransformed into yeast strain AH109,respectively.The transformed AH109 could grow well in auxotroph medium SD-Trp-Leu-DO,but could not grow in auxotroph medium SD-Trp-Leu-Ade-His-DO,and both the reporter gene MEL1 and LacZ could not be activated.All these results showed that there was no direct interaction between OsRUS1and OsRUS2.1.