花鲈TYR基因表达和功能分析

Expression and Function Analysis of TYR from Lateolabrax maculatus

花鲈(Lateolabrax maculatus)是中国重要的海水鱼,它可以在河口或淡水中生长。体色是鱼类重要的生物学特征,与鱼或鱼肉的外观有密切关系,影响其市场价值。酪氨酸酶(tyrosinase,TYR)是黑色素合成的关键酶。本研究克隆了花鲈TYR基因,其包括完整开放阅读框1620 bp,编码539个氨基酸,分子量为61.1 kD,理论等电点为6.04。花鲈TYR有19个氨基酸编码的信号肽,一个跨膜结构域。花鲈TYR基因还含有2个保守的结构域:EGF-like结构域和铜离子结合区域,还有6个保守的组氨酸位点。系统进化分析表明花鲈TYR与大黄鱼(Larimichthys crocea)有着较高的同源性,物种间亲缘关系与其传统分类地位一致。蛋白质结构预测也表明花鲈TYR的三维结构具有典型的酪氨酸酶特征,包括酪氨酸结构域和铜离子结合的氨基酸位点。PCR结果显示花鲈TYR主要在鳃、脾、皮肤、胃盲囊、中肾、头肾和伪鳃中表达。在胚胎期发育中,TYR从原肠期开始表达。本研究结果说明花鲈TYR不仅和黑色素的合成有关,也可能参与其它生理活动以及花鲈早期胚胎发育,为进一步研究鱼类TYR的功能和进化提供理论依据。

Lateolabrax maculatus is an important marine fish in China.It can grow in estuary and fresh water.Body color is an important biological feature of fish,it is also closely related to the appearance of live fish or fish meat product,and affected its market value.Tyrosinase(TYR)is the key enzyme in melanin synthesis.The L.maculatus TYR(Lm-TYR)gene was cloned and analyzed in this study.The gene includes 1620 bp open reading frame,coding 539 amino acid.The protein has a predicted molecular mass of 61.1 kD and theoretical isoelectric point is 6.04.Lm-TYR has a 19 amino acids putative signal peptide at the N terminal and a transmembrane region at the C-terminal end.The Lm-TYR gene family contains two conserved domain:EGF-like domain and copper ion binding domain.The protein has 6 conserved histamine sites.Phylogenetic analysis showed that the Lm-TYR gene was classified to a subfamily of teleost with high homology with Larimichthys crocea. The genetic relationshipbetween species is consistent with their traditional taxonomic status. Protein structure prediction indicated that thethree-dimensional structure of gastric caecum has typical TYR protein characteristics, including tyrosine domainand two copper ion-binding sites. RT-PCR showed that Lm-TYR was mainly expressed in gill, spleen, skin, gastriccaecum, kidney, head kidney and pseudobranch. During embryonic development, Lm-TYR gene is expressed fromthe gastrula stage. The results showed that the tyrosinase was not only involved in the synthesis of melanin, butalso involved in other physiological activities and early embryonic development. This paper lays a foundation forfurther research on the function of fish TYRs.