Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine
CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the
CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes,
CAST-1,
CAST-2 and
CAST-6 to
CAST-11, were identified. Haplotypes
CAST-1 and
CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype
CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98–99%, 94–98%, 92–93% and 82–83% similarity to the published ovine, caprine, bovine and porcine
CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A > T, c.282G > T and c.296C > G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in
CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine
CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2 + channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine
CAST locus (0.802). Frequency difference of haplotypes
CAST-10 and
CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly significant (P < 0.001), indicating that these two haplotypes might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds.
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