Sequence Analysis Of 22 Kda-like α-coixin Genes And Their Comparison With Homologous Zein And Kafirin Genes Reveals Highly Conserved Protein Structure And Regulatory Elements

Several genomic and cDNA clones encoding the 22 kDa-like α-coixin, the α-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like α-coixin genes designated α-3A, α-3B and α-3C were found in the 15 kb α-3 genomic clone. The α-3A and α-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the α-3B gene, suggesting that the three α-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of α-coixin clones with the published sequences of 22 kDa α-zein and 22 kDa-like α-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like α-prolamins and the 19 kDa α-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5′ and 3′ flanking regions of α-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in α-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in α-3B that occupies approximately the same positions as those identified for the 22 kDa α-zein and α-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like α-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation. © 1993 Kluwer Academic Publishers.21576577