西里伯斯青鳉tyr和slc24a5的克隆及表达分析

为了探究黑色素合成相关基因酪氨酸酶(Tyrosinase,tyr)和溶质载体24家族成员5(Solute carrier family 24,member 5,slc24a5)在西里伯斯青鳉(Oryzias celebensis)中的表达特征,研究采用RACEs(Rapid Amplication of cDNA En...

CLONING AND EXPRESSION ANALYSIS OF TYR AND SLC24A5 IN ORYZIAS CELEBENSIS

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.