半滑舌鳎(Cynoglossus semilaevis)黑色素富集激素基因的克隆和表达

Cloning and Expression of Melanin-Concentrating Hormone in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

中文关键词: 半滑舌鳎  黑色素富集激素  无眼侧黑化  表达调控

英文关键词: Cynoglossus semilaevis  Melanin-concentrating hormone  Hypermelanosis on the blind-side  Expression regulation

中文摘要:

      为研究黑色素富集激素基因(MCH)表达调控与半滑舌鳎(Cynoglossus semilaevis)无眼侧黑化性状的关系，通过分子克隆获得了半滑舌鳎pMCH2，通过NCBI获得了pMCH1 cDNA序列，分析了pMCH mRNA的组织表达特性，探索了脑垂体和皮肤中的pMCH mRNA表达与无眼侧黑化程度的关系。结果显示，pMCH1 cDNA序列长476 bp，编码134个氨基酸，与鲽形目、鲤形目和鲈形目鱼类的pMCH1氨基酸聚为1个进化分支。pMCH2 cDNA序列全长为626 bp，编码147个氨基酸，与鲽形目和鲀形目鱼类的pMCH2氨基酸聚为1个进化分支。2种MCH mRNA在垂体中表达量最高，同时，MCH1 mRNA在除肌肉外的其他组织中均可检测到表达；MCH2 mRNA在脑、有眼侧皮肤、无眼侧正常皮肤、性腺和鳃组织中也可检测到较高表达。MCH mRNA表达与无眼侧黑化程度的关系分析显示，脑垂体和皮肤中MCH1 mRNA表现出相似的表达变化趋势，都是10%黑化组的表达量最高，而后随黑化程度加大显著降低。对于MCH2而言，无眼侧正常鱼和无眼侧50%黑化鱼的脑垂体中都具有较高的MCH2 mRNA表达水平，但在无眼侧10%黑化组和无眼侧80%黑化组，其表达水平显著降低。皮肤中MCH2表现出随黑化程度加大而显著升高的趋势。本研究结果可为认识MCH基因与半滑舌鳎无眼侧黑化性状的表达调控关系提供基础资料。

英文摘要:

      Hypermelanosis is an abnormal coloration that commonly occurs on the blind-side of half-smooth tongue sole Cynoglossus semilaevis in captivity, resulting in deterioration in flesh quality and reduced market price. This problem has become the bottleneck for sustainable development of flatfish farming industry. Melanin-concentrating hormone (MCH) is produced in pituitary acting as an inhibitor for hypermelanosis in fish. The present study aims to identify the role of MCH in regulating the blind-side hypermelanosis in C. semilaevis. cDNAs encoding two MCH alleles (pMCH1 and pMCH2) were cloned using RACE method and their structures were analyzed. The spatial and temporal expression patterns of MCH mRNA were measured using the quantitative real-time PCR. Results showed that C. semilaevis pMCH1 cDNA sequence was 476 bp in length and encoded 134 amino acids, which shares high identity (66.9%) with Paralichthys olivaceus. C. semilaevis pMCH1 was clustered with the Pleuronectiformes, Tetraodontiformes and Perciformes species based on the phylogenetic analysis. By contrast, C. semilaevis pMCH2 cDNA sequence was 626 bp in length and encoded 147 amino acids. The transcript levels of both MCH1 and MCH2 had the highest expression level in pituitary compared to other tissues. MCH1 mRNA was also detected among other tissues. Furthermore, MCH2 mRNA was highly expressed in the brain, eye-side skin, blind-side skin, gonad and gill, whereas the peripheral tissue had relatively low expression level. Correlating the MCH mRNA expression levels and the degree of the blind-side hypermelanosis showed that, MCH1 mRNA expression levels in the pituitary and skin had similar trends, which peaked when fish had about 10% blind-side hyperpigmentation, and then significantly reduced as the blind-side hypermelanosis level increased. For fish with normal blind-side coloration and 50% blind-side hyperpigmentation, their pituitary MCH2 mRNA levels were significantly higher than those from fish with 10% and 80% blind-side hyperpigmentation. The skin MCH2 mRNA level significantly increased with the increased hyperpigmentation degree on the blind-side of fish. The present study provides new insights into the mechanisms underlying the regulation of blind-side hypermelanosis in C. semilaevis.