Identification of a DNA-binding factor that recognizes an alpha-coixin promoter and interacts with a Coix Opaque-2 like protein

Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the r egulation of expression of a 22 kDa zein-like alpha-coixin gene. A set of u nidirectional deletions was generated in a 962 bp fragment of the alpha-coi xin promoter that had been previously fused to the reporter gene GUS. The c onstructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from -1084 to -238. However, deletion from -238 to -158, which partially de leted the O2c box, resulted in a dramatic decrease in GUS activity emphasiz ing the importance of the O2 box in the quantitative expression of the gene . The -238 promoter fragment interacted with Coix endosperm nuclear protein s to form 5 DNA-protein complexes, C1-C5, as detected by EMSA. The same ret arded complexes were observed when the -158 promoter fragment was used in t he binding reactions. Reactions with nuclear extracts isolated from Coix en dosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the alpha-coixin promoter are diff erentially assembled during seed development. Deletion analysis carried out on the -238/ATG promoter fragment showed that a 35 bp region from -86 to - 51 is essential for the formation of the complexes observed. When nuclear e xtracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented ind icating that an Opaque-2 like protein participates in the formation of thos e complexes. Complex C2 was not affected by the addition of the O2 antibody , suggesting the existence of a novel nuclear factor, CBF1, that binds to t he promoter and makes protein-protein associations with other proteins pres ent in Coix endosperm nuclei.