Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic rice seeds

A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to Triticum aestivum puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to -1068, -388, - 210 or -124 upstream of the translation initiation ATG codon and the sequen ce coding for the first 13 amino acids of the puroindoline-b, were translat ionally fused to the uidA reporter gene encoding beta-glucuronidase and tra nsferred to rice calli via particle bombardment-mediated transformation. Th e 1068 bp and 124 bp promoters were also transcriptionally fused to the uid A reporter gene. Out of the 196 plants regenerated from transformed rice ca lli, 118 plants set seeds. No GUS activity was detectable in the stems, roo ts, leaves or pollen of the transgenic rice which had integrated the puroin doline-b promoter or its deletions; GUS activity was detected only in seeds , except in those having integrated the 124 bp promoter. Within seeds, hist ological localisation showed GUS activity as being restricted to the endosp erm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5' promoter deletions identified the reg ion between -388 and -210 as essential for endosperm expression, and the re gion between -210 and -124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the transl ational and transcriptional fusion genes.