Abstract:

The aim was to explore the effective situs of starch branching enzyme between its interactive protein in Zea mays and to construct bait vectors in yeast two-hybrid. The amino acid sequence of SBEIIb protein was analyzed by the CDD and NCBI, and the functional domain and non-functional domain were determined. The genes of the functional domain and non-functional domain were amplified by PCR, and purified and cloned into the pGBKT7 plasmid after digested with the same restriction endonuclease. The pGBKT7-SBEIIb-X (X=1, 2, 3, 4, 5, 6) of the yeast two-hybrid bait vector were successfully transformed into the yeast cells AH109, and OD600 value over 0.8. The result indicated that bait vectors were not toxic to AH109 cells. The yeast transformants grew white colonies in SD/-Trp/X-α-Gal plates, but couldn't in SD/-His/-Trp/X-α-Gal and SD/-Ade/-Trp/X-α-Gal plates, so except for self-activation of the bait protein. Vectors of pGBKT7-SBEIIb-X (X=1, 2, 3, 4, 5, 6) were constructed, and were not toxic to AH109 and could not activate the reporter genes, which would provide a foundation for further analyzing the effective situs of interactive protein with SBEIIb.