Nucleotide sequence and endosperm-specific expression of the structural gene for the thionin alpha-hordothionin in barley (Hordeum vulgare L.)

Abstract

A barley genomic library, obtained by cloning in the vector λEMBL-4, was screened with a cDNA probe encoding the α-hordothionin toxin. A positive clone, designated λTH1, was selected for further characterization. The coding and flanking regions of the α-hordothionin gene (Hth-1) were sequenced. Hth-1 has two introns of 420 and 91 nucleotides (nt), respectively. The promoter region has the following main features: one TATA box; three CATC boxes; an enhancer-like sequence, starting at nt position −282 from the first ATG codon, which is homologous to sequences appearing at similar positions in other endosperm genes; two versions of an 18-nt sequence that is more highly repeated in structural domains of several prolamin genes; two extensive regions close to the first ATG codon that are homologous to a sequence located much further upstream in the B-hordein promoter. The transcription start point was determined at nt positions −46 to −47, both by the S1 nuclease-protection and by the primer-extension assays. A maximum of 2–4 copies of the Hth-1 gene per haploid genome was determined by Southern-blot hybridization. Expression of the Hth-1 gene was detected during the cell proliferation stage of endosperm development (maximum at 13–16 days after pollinization) and was not detected in either etiolated or green coleoptiles