, Volume 12, Issue 3, pp 245-256

Characterization of the kafirin gene family from sorghum reveals extensive homology with zein from maize

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Abstract

Electrophoretic analysis of translation products of polyadenylated RNA isolated from mid-maturation sorghum seed in the presence of [35S]met, [3H]leu, or [3H]val revealed two major proteins of kDa and 21 kDa. These products were not detected when [3H]lys was supplied as the radioactive substrate. Under similar electrophoretic conditions, kafirin (a major seed storage prolamin of sorghum), migrated as two bands of 22 kDa and 19 kDa. Sequence analysis of two cDNA clones (pSK8 and pSKR2) from sorghum seed mRNA revealed them to be highly homologous with each other and to the 22 kDa zeins from maize, suggesting that they represented kafirin cDNAs. Compared with pSKR2, pSK8 had an insertion of 24 nucleotides and a deletion of 24 nucleotides, so that the coding regions were nearly identical in length. The deduced amino acid sequence for these cDNA clones reveals that kafirin, like zein, is rich in glutamine and nonpolar amino acids, but contains no lysine. Both kafirin and zein have a 21 amino acid signal peptide exhibiting 80% homology and eight copies of a repetitive amino acid block in the C-terminal domain with the consensus: infI supP LL finP supA LN infQ supP LALANPAAYLQQQQ.

The kafirin cDNAs were used as probes to screen a sorghum genomic library; one genomic clone (λGK.1) was sequenced and found to be very similar (97.8%) to the pSK8 cDNA clone. Clone λGK.1 contains features typical for a functional gene in that the intronless open reading frame encoding 268 amino acids is flanked at the 5′ end by sequences corresponding to the CAAT and TATA promoter boxes (positioned at about −60 and −30 bp, respectively, from the transcriptional initiation site), and at the 3′ end by a consensus polyadenylation signal. In common with zein genomic clones, kafirin clones contain a 15 basepair consensus sequence centered at postion −320 relative to the transcriptional initiation site. Under similar hybridization conditions, genomic reconstruction analysis using an oligonucleotide probe indicated the presence of less than 20 copies of kafirin per haploid sorghum genome compared with approximatley 140 copies of zein per haploid maize genome.