Yeast Two-Hybrid Screening of Maize and Wheat NF-YB Factors

The CDS of ZmNF-YB2a (accession no. NP_001105435) was cloned into the vector pGBKT7 (Invitrogen, Victoria, Australia) for Y2H screening. ZmNF-YB2a was used because maize ZmNF-YB2 cDNA could not be isolated based on the sequence submitted to public databases by Nelson et al. (2007) . The only protein sequence difference between the two is an insert of seven amino acid residues (GKTIPAN) in the HFD of ZmNF-YB2, which is absent in ZmNF-YB2a and similar NF-YB subunits from wheat and other grasses. ZmNF-YB2a was used as bait to screen WGL and WENDL (Lopato et al., 2006 (link)), WHSL (Eini et al., 2013 (link)), WGD (developing grain at 0–6 days after pollination collected from the wheat cv. RAC875 subjected to drought at flowering), and WRDL (roots of wheat cv. RAC875 seedlings grown in soil and subjected to drought) cDNA libraries as previously described (Eini et al., 2013 (link)). A large number of independent clones containing full-length or partial coding regions of the TaNF-YC15 cDNA were isolated. Because of self-activating properties of the full-length TaNF-YC15, a truncated version of the protein beginning at residue Ala97 was used to re-screen the same cDNA libraries. Clones containing inserts encoding two different full-length NF-YB TFs, designated as TaNF-YB2 (18 independent clones) and TaNF-YB4 (22 independent clones), were isolated.