STRUCTURAL AND FUNCTIONAL-ANALYSIS OF PROMOTER FROM GLIADIN, AN ENDOSPERM-SPECIFIC STORAGE PROTEIN GENE OF TRITICUM-AESTIVUM L

To identify cis-regulatory elements of the gliadin gene, a study of the gliadin gene promoter was conducted by transient expression analysis of plasmid DNAs which were introduced into plant protoplasts by electroporation. The promoter region (- 592 bp to + 18 bp from the translational start) of this developmentally regulated gene, when fused upstream to the chloramphenicol acetyl transferase (CAT) reporter cassette was unable to direct significant CAT expression in wheat or tobacco suspension cells. Because this monocot gene promoter appeared to be under stringent tissue-specific control, a hybrid promoter approach using a nopaline synthatase (nos) promoter was employed. A series of 3' deletions of the gliadin promoter were placed upstream of either a nonfunctional -101 nos or a nearly wild-type -155 nos promoter fused in turn to a CAT reporter gene cassette. Transient expression analysis of these plasmid DNAs in tobacco cells showed that the gliadin fragment could either restore the activity of the non-functional nos promoter (series I) or enhance the activity of the functional nos promoter (series II). The degree of restoration of the promoter function conferred by gliadin fragments of the first series was proportional to the enhancing effect of the same fragments in the second series of constructs. The transcriptional activity of the gliadin (-592 bp to -77 bp)-nos hybrid promoter was reduced by 26% upon 3' deletion of sequences in the region -141 bp to -77 bp, which contains both the TATA and CCAAT boxes. A marked decline in the promoter function of these hybrid constructs, however, was observed when an additional upstream region was removed, suggesting the presence of regulatory sequences in the -218 bp to -141 bp region of the gliadin promoter. Deletion of the -300 bp element, which is similar to the SV40 core enhancer, did not affect hybrid promoter function, although additional upstream activating sequences (-592 bp to -448 bp) were also observed.