Construction of OsRus1 Yeast Two-Hybrid Bait Vectors and Detection of Their Self-activated Activity
Four fragments of rice(Oryza sativa L)ROOT UV-B SENSITIVE 1(OsRUS1),OsRUS1(1-1782),OsRUS1(1-504),OsRUS1(510-1282),OsRUS1(1188-1782),were amplified by PCR from cloned OsRUS1,and were ligated with pMD18-T-Simple,then transformed to E.coli TOP10 competent cell.The positive clones were selected and sequenced.The confirmed fragments were subcloned to bait vector pGBKT7.The four constructed pGBKT7 bait vectors were further confirmed by enzyme digestion and sequencing.The confirmed pGBKT7 bait vectors were transformed to Y187 yeast competent cell.The self-activation and toxicity of the plasmids to host yeast Y187 by LacZ and MEL1 activity assays and culturing in auxotroph medium SD-Trp-DO.Results showed that the four constructed plasmids had no self-transcriptional activity and not toxicity to yeast strain Y187.The four bait vectors constructed could be used in yeast two hybrid system,which laid the foundation for screening interactional proteins of OsRUS1 from rice cDNA library.