半滑舌鳎(Cynoglossus semilaevis)体表色素细胞观察及POMC表达特性分析

摘要

The hypermelanosis on the blind-side ofCynoglossus semilaevis have reduced its market value and affected its availability in the enhancement program, which has adverse effects on the sustainable development of the farming industry. However the underlying mechanisms of hypermelanosis have not been clarified yet. To investigate the cytological mechanism of hypermelanosis on the blind-side ofC. semilaevis, we identified three kinds of chromatophores, namely melanophore, xanthophore and iridophore, based on the microscopic observation, and we described their morphology. The melanophore contained black and brown pigment granules and had a relatively larger nucleus. They could be characterized into two types according to the morphology. In contrast, the nuclei of xanthophore and iridophore were relatively small. The distribution and pattern of the three chromatophores in the eye-side and the blind-side skin were examined and compared. We isolated and sequenced the proopiomelanocortin (POMC) cDNA from pituitary using RT-PCR and RACE. The POMC cDNA was 910 bp in length consisting of a 114 bp 5'-UTR, a 154 bp 3'-UTR, and a 642 bp open reading frame that encoded 213 aminoacids. The amino acid sequence was composed of the signal peptide, ACTH,α-MSH,βMSH,γ-LPH, andβ-endorphin domains, withoutγ-MSH and most of the joining peptide domains. The amino acid homology analysis revealed thatC. semilaevis POMC shared 30%?64% similarity with their fish counterparts. Quantitative PCR results showed that POMC mRNA was mainly expressed in the pituitary and was also expressed in the brain, gonad and the black skin (hypermelanosis) on the blind side. The POMC mRNA expression level in the blind-side black skin was significantly higher than that in the ocular-side and in the blind-side normal skin, which indicated a close correlation between the POMC gene and the hyperpigmentation on the blind-side. Our study provided basic information on the cytological and molecular mechanisms of hyperpigmentation on the blind-side ofC. semilaevis.%为认识养殖半滑舌鳎无眼侧黑化的细胞学特性,利用显微观察方法研究了其皮肤黑色素细胞、黄色素细胞和虹彩细胞的形态特征,比较了3种色素细胞在有眼侧皮肤、无眼侧正常和黑化皮肤中的数量分布特征.为进一步揭示无眼侧黑化的分子机制,克隆了半滑舌鳎POMC基因的cDNA序列.结果显示,黑色素细胞较大,含黑色和棕色的色素颗粒,有树突状分枝不明显和延伸成放射状两种形态;黄色素细胞较小,含黄色素颗粒;虹彩细胞最小,含鸟粪素颗粒.半滑舌鳎 POMC基因的cDNA序列长910 bp,包括一个114 bp的5'非翻译区和一个154 bp的3'非翻译区,开放阅读框长度为642 bp,共编码213个氨基酸,包含ACTH、α-MSH、β-MSH、γ-LPH、β-内啡肽5个多肽序列,但缺失γ-MSH和大部分连接区.半滑舌鳎POMC基因的氨基酸序列与其他鱼类的同源性为30%-64%.定量PCR分析显示,POMC mRNA主要在垂体中表达,其次是脑、性腺和无眼侧黑化皮肤.正常与黑化皮肤中的差异表达结果显示,无眼侧黑化皮肤中POMC mRNA表达量最高,并与有眼侧皮肤和无眼侧正常皮肤中的POMC mRNA表达量差异显著,揭示了POMC的表达与无眼侧黑化性状密切相关.