Construction of a high-quality yeast two-hybrid (Y2H) library and its application in identification of interacting proteins with key vernalization regulator TaVRN-A1 in wheat

BACKGROUND: Low temperature is required for the competence of winter wheat to flowering (vernalization), and several key components in the vernalization-mediated flowering pathway have been isolated. A Y2H library is a very useful platform to further unravel novel regulators in the flowering pathway. Thus, there is a necessity to construct a high-quality Y2H library using vernalized winter wheat plants. RESULT: We described the construction of a high-quality Y2H library using winter wheat plants with cold-treatment for different weeks to maximize pooling interacting proteins during vernalization. The resultant Y2H library contained ~2.5×10(6) independent clones, with a cell density of ~2.6×10(8) and an average insert size of ~ 1.5 kb. TaVRN-A1 was used as a “bait” to test the quality of the Y2H library. As a result, several cDNA clones encoding TaSOC1 and TaSVP1 that were known to have a direct binding with TaVRN-A1 were identified, demonstrating that the Y2H screen system constructed in this study was highly efficient. Additional proteins that were discovered but not characterized in previous studies could be novel partners of TaVRN-A1 in wheat. CONCLUSION: We established a high-efficient Y2H screen system using the Matchmaker™ technology with several modifications in the critical steps. Ultimately, we provided a successful example to fast and economically create high-quality Y2H libraries for studies on protein interaction in hexaploid wheat.