Zhou 2016

Introduction to KAP6 Gene Family

  • KAP6 is part of the high glycine-tyrosine keratin-associated protein (HGT-KAP) family.

  • This family is believed to encompass multiple genes, and the study identifies new members in sheep.

Identification of New KAP6 Genes

  • A KRTAP6 coding sequence was utilized to search the Ovine Genome (v3.1).

  • Five homologous regions (R1–R5) were detected, each containing an open reading frame (ORF).

  • Regions R1–R5 showed high similarity to sheep skin Expressed Sequence Tags (ESTs).

  • Phylogenetic analysis classified R1–R5 with KAP6 sequences from various species, separate from other HGT-KAPs.

Characteristics of Regions

  • R1: Similar to characterized KRTAP6-1.

  • R2–R5: New sequences, distinct from known KAPs.

  • PCR primers confirmed the presence of these genes, producing amplicons in 96 sheep.

  • SNP Variations:

    • KRTAP6-2: 5 SNPs observed.

    • KRTAP6-3: 3 SNPs and a 45-bp insertion/deletion detected.

    • KRTAP6-4: 3 SNPs.

    • KRTAP6-5: 5 SNPs and an 18-bp insertion/deletion.

  • Allele frequencies varied significantly between Merino and Romney breeds.

Role of Keratin-Associated Proteins (KAPs)

  • KAPs are crucial for wool and hair fibre structures, forming a semi-rigid matrix for keratin filaments.

  • Compositions mainly consist of cysteine, glycine, and tyrosine.

  • KAPs are categorized into three groups:

    • High Sulfur (HS) KAPs

    • Ultra-High Sulfur (UHS) KAPs

    • High Glycine-Tyrosine (HGT) KAPs

  • Presence in wool varies, influencing fibre properties and characteristics.

Variation and Polymorphism in KAP6 Genes

  • Study aimed to explore variations in newly identified KAP6 genes.

  • Four KAP6 genes identified showed polymorphism across different breed samples.

  • KRTAP6 genes established have similar coding sequences but differ significantly in flanking regions, impacting gene expression.

Phylogenetic Analysis and Comparisons

  • R1–R5 genes clustered closely with known KAP6 sequences from various species.

  • Suggested that KAP6 gene family is more diverse in sheep than in other species, such as humans (three members).

Experimental Validation

  • PCR experiments confirmed the presence of four new KAP6 genes using region-specific primers.

  • SSCP analysis identified multiple allele patterns, showing variant sequences:

    • R2: six alleles with five SNPs (all synonymous).

    • R3: five alleles, including a 45-bp insertion/deletion.

    • R4: three alleles, one non-synonymous SNP.

    • R5: identified six alleles with specific insertions/deletions.

Genetic Variation Between Breeds

  • Significant genetic variance identified between NZ Romney and Merino sheep

  • KRTAP6-3: allele B prevalent in Merino, absent in NZ Romney.

  • KRTAP6-4: allele C common in NZ Romney, rare in Merino.

  • KRTAP6-5: allele D found more frequently in Merino than NZ Romney.

Discussion and Implications

  • Discovery of four new KAP6 genes increases known family members from one to five.

  • This supports previous studies indicating multiple KAP6 genes exist in sheep.

  • Identified genes may link to wool traits and potential genetic markers for breeding.

  • More extensive studies needed to define gene impact on wool characteristics.

Methodology Overview

  • Research adhered to Animal Welfare legislation for sheep blood collection.

  • 96 sheep samples analyzed (48 Merino, 48 NZ Romney).

  • Genomic DNA isolated and analyzed through established protocols, including PCR amplification and SSCP for heterozygote detection.

  • Allelic variants sequenced for genetic variability assessment.

Conclusion

  • Revealed a substantial diversity in KAP6 genes with implications for understanding wool genetics and improvement through breeding strategies.

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