西里伯斯青鳉tyrslc24a5的克隆及表达分析

CLONING AND EXPRESSION ANALYSIS OF TYR AND SLC24A5 IN ORYZIAS CELEBENSIS

  • 摘要: 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 Ends)技术从西里伯斯青鳉精巢中克隆了tyr(Octyr)和slc24a5(Ocslc24a5)的全长cDNA(Complementary DNA)。Octyr的cDNA全长为2249 bp, 开放阅读框长1635 bp, 编码545个氨基酸; Ocslc24a5的cDNA全长为2065 bp, 开放阅读框长1542 bp, 编码514个氨基酸。氨基酸多重比对和系统发育树分析发现, OctyrOcslc24a5氨基酸序列非常保守, 均与日本青鳉同源性较高。通过PCR克隆了OctyrOcslc24a5的基因组DNA序列, 结果发现, Octyr开放阅读框跨越5个外显子, Ocslc24a5开放阅读框跨越8个外显子。荧光定量PCR(qRT-PCR)分析了OctyrOcslc24a5在不同组织和不同胚胎发育时期的相对表达水平, 结果显示, OctyrOcslc24a5在脑、眼、肝、肾、肠、皮肤、卵巢和精巢中均有表达, 且均在精巢中表达量最高, 其次是眼和脑; OctyrOcslc24a5在胚胎发育不同时期均有表达, 在胚胎发育到第7天时表达水平均达到最高。利用0.25 mmol/L苯基硫脲(PTU)抑制胚胎黑色素沉积后进行整体原位杂交(WISH)发现, OctyrOcslc24a5的表达模式相似, 在囊胚期, OctyrOcslc24a5在所有胚胎细胞中均有表达; 在神经胚期, OctyrOcslc24a5主要在背部和卵黄膜中表达; 在体节期, OctyrOcslc24a5 在眼睛、脑和背侧皮肤中表达。以上研究结果说明OctyrOcslc24a5可能参与西里伯斯青鳉黑色素的合成, 研究为深入了解西里伯斯青鳉黑色素形成的分子机制奠定基础。

     

    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.

     

/

返回文章
返回