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 (21-2 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.Barley endosperm contains two chymotrypsin inhibitors called CI-1 and CI-2 which belong to a family of proteins including the potato inhibitor I and the leech inhibitor elgin [l]. CI-2 normally accounts for about 0.25% of the total salt-soluble proteins of the barley grain (calculated from [2]). However, the spontaneous high-lysine barley mutant Hiproly [3] has a 20-fold increased content of CI-2 [4, 51. Because of its high lysine content (11.5 g YO), CI-2 accounts for 37% of the difference in lysine content between Hiproly and normal barley grains and so contributes significantly to the improved nutritional quality of Hiproly. The protein has therefore received considerable attention. It has been purified from Hiproly endosperms and characterized in detail [5 -71.The increased amount of CI-2 in Hiproly is controlled by a single recessive gene called lys on chromosome 7 [3, 81, whereas the structural genes for CI-2 are located at the Zca-2 locus on chromosome 5 [9]. It is not known how the product of the lys gene affects the regulation of expression of the Zca-2 locus, though a recent study by Rasmussen [lo] suggests that the increased amount of CI-2 in Hiproly may stem from an earlier synthesis of CI-2 during endosperm de...

A plant serpin gene

“…4B) and the synthesis of an immunologically related polypeptide encoded by a locus on chromosome 7 [9] [33] and thionins [36] are all synthesized in the developing endosperm and are coded for by small multigene families. Of these the Hor2 locus on chromosome 5, the gene family encoding B-hordein polypeptides, has been studied in detail.…”

“…5). Among the chymotrypsin inhibitors, CI-2 is synthesized without a cleavable signal peptide, but has an internal hydrophobic sequence [33] and is transported via the Golgi apparatus to the vacuole of the cell [34]. Like protein Z4, P-amylase is synthesized in the mature form [lo] on membrane-bound polysomes [I 61.…”

Opportunities for manipulating the seed protein composition of wheat and barley in order to improve quality

“…Note the high content of lysine residues (K) and absence of cysteine. Based on data in Williamson et al (1987Williamson et al ( , 1988. Standard single letter abbreviations for amino acids are used.…”

Three cDNA clones for barley leaf acyl carrier proteins I and III

“…Since no other experimental evidence is available to support that this protein is an ACP, it is suggested that henceforth the name ACP IlI be used for the pACP 1 coded protein. Figure 4 reveals that the poly A signal (38) at bases 656-661 (numbers correspond to pACP 11 in Figure 4), in contrast to those in the other two cDNAs, is followed 56 bases later by a tail of at least 51 adenines. The open reading frame translating into 49 amino acids in the 3' region lacks the consensus initiation sequence.…”