Abstract:
Melanin is widely distributed in animals and is one of the important factors in the formation and maintenance of body color. Many studies have shown that
tyr and
slc24a5 play crucial roles in the synthesis of melanin in vertebrates.
tyr is the key rate-limiting enzyme in melanogenesis. It catalyzes and oxidizes the precursor tyrosine to form dopa-quinone, eumelanin and pheomelanin after a series of complex regulation.
slc24a5 is the fifth member of the solute carrier 24 family, and the protein encoded by NCKX5, an important pigmentation gene in human. To explore the expression patterns of melanin-related genes
tyr and
slc24a5 in
O. celebensis, the full-length cDNAs of
O. celebensis tyr (
Octyr) and
slc24a5 (
Ocslc24a5) were cloned by rapid amplification of cDNA ends (RACEs). The full length of
Octyr gene was 2249 bp, including a 126 bp 5′ untranslated region (UTR), a 488 bp 3′UTR and a 1635 bp CDS for 545 amino acids. The full length of
Ocslc24a5 gene was 2065 bp, including a 160 bp 5′UTR, a 363 bp 3′UTR and a 1542 bp open reading frame for 514 amino acids. Multiple alignments of amino acid and phylogenetic tree analysis revealed that the amino acid of
OcTyr and
OcSlc24a5 were conserved, and they had high homologies with Japanese medaka. Genomic sequence of
Octyr and
Ocslc24a5 were obtained by PCR cloning. A total of 6.3 kb of the Octyr genome with 5 exons was cloned. The genome length of
Ocslc24a5 was 6.08 kb. Interestingly, we found that the genome of
Ocslc24a5 has eight exons and seven introns, while zebrafish, human and mouse have nine exons and eight introns. qRT-PCR showed that
Octyr and
Ocslc24a5 were expressed in brain, eye, liver, kidney, gut, skin, testis, and ovary, with the highest expression level in testis, followed by eye and brain. Both
Octyr and
Ocslc24a5 were abundant during embryonic development, with the highest expression level at the seventh day’s embryo. In order to study the spatiotemporal expression pattern of
Octyr and
Ocslc24a5 in the development of embryos, whole mount
in situ hybridization (WISH) assay was conducted. WISH indicated that the expression patterns of
Octyr and
Ocslc24a5 were similar.
Octyr and
Ocslc24a5 were detected in all embryonic cells at the blastula stage.
Octyr and
Ocslc24a5 were concentrated on the back and yolk membrane at the neurula. During the somitogenesis stage,
Octyr and
Ocslc24a5 were abundant in eye, brain and dorsal skin. In summary,
Octyr and
Ocslc24a5 may be involved in the synthesis of melanin in
O. celebensis. This study provides important information for studying body color formation and lays a foundation for understanding the molecular mechanism of melanin formation in
O. celebensis.