Nucleotide sequence of barley chymotrypsin inhibitor‐2 (CI‐2) and its expression in normal and high‐lysine barley

Abstract

cDNA clones for chymotrypsin inhibitor-2 (CI-2) have been isolated from an endosperm-specific library of barley using a synthetic oligonucleotide probe. The nucleotide sequences of several of the cDNAs predict an open reading frame (beginning with an ATG codon) which encodes a protein of 84 residues (Mr 9380). In the longest clone another ATG codon is present, a further 69 nucleotides upstream. The nucleotide sequence between these two ATG codons predicts an amino acid sequence with the characteristics of a signal peptide, as found in other cloned plant protease inhibitors. However, it contains an in-frame TAA stop codon, which is also present in all of the shorter cDNAs which extend into this region. From in vitro translation experiments, using mRNAs synthesized from cDNAs, we conclude that, in vitro, translation of all or the vast majority of CI-2 mRNAs begins at the second ATG codon, 31 nucleotides downstream from the ochre stop codon. Southern blotting of genomic DNA shows that CI-2 is encoded by a small multigene family, while sequence analysis of the cDNAs shows that at least two sub-families of mRNAs, which are more than 90% homologous, are present in the endosperm. Northern blotting analysis shows that related but different sequences are present in leaf and shoot RNA populations. Further Northern blot hybridizations using RNA from the normal line, Sundance, and the high-lysine barley mutant, Hiproly, show that endosperms of the latter contain greatly increased levels of CI-2 mRNA. This correlates with the increased amount of CI-2 protein deposited in Hiproly, and demonstrates that the differential expression of CI-2 in the two genotypes is controlled at the level of transcription and/or stability of the mRNA. In contrast, the abundance of CI-2 mRNAs in leaves and shoots is not affected.