文章摘要
锦鲤墨蝶呤还原酶基因(sepiapterin reductase, spr)的克隆、表达和定位分析
The molecular cloning and expression analysis of sepiapterin reductase in Japanese ornamental carp (Cyprinus carpio var. koi)
投稿时间:2019-07-07  修订日期:2019-10-12
DOI:
中文关键词: spr  锦鲤  克隆  碟啶代谢  体色
英文关键词: spr  koi carp  cloning  pteridines metabolism  color formation
基金项目:国家自然科学基金青年科学基金 (31402294),河南省重点研发与推广专项(182102110164, 192102110192),农业部休闲渔业重点实验室开放基金课题 (2019N06)
作者单位E-mail
胡菊 河南师范大学水产学院 2440187244@qq.com 
冯彩 河南师范大学水产学院  
马晓 河南师范大学水产学院  
吴利敏 河南师范大学水产学院  
刘慧芬 河南师范大学水产学院  
宋红梅 中国水产科学研究院珠江水产研究所  
胡隐昌 中国水产科学研究院珠江水产研究所  
田雪 河南师范大学水产学院
中国水产科学研究院珠江水产研究所 
tianxue_81@126.com 
李学军 河南师范大学水产学院  
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中文摘要:
      鱼类体色来源于皮肤和鳞片中分布的色素细胞,色素细胞具有的色素颗粒可分为黑色素、类胡萝卜素、蝶啶和嘧啶四种成分。墨蝶呤是一种黄色素蝶呤,经墨蝶呤还原酶(sepiapterin reductase, SPR)及二氢叶酸还原酶(dihydrofolate reductase, DHFR)催化后生成四氢生物蝶呤(tetrahydrobiopterin, BH4),最终生成黄色/红色的蝶啶色素颗粒。SPR是BH4从头合成途径中最后一步催化酶。为探究SPR在锦鲤体色形成中的作用,本研究利用RACE技术获得spr cDNA全长序列,并分析其时空表达模式,同时利用Western blot和免疫组织化学方法检测SPR蛋白在皮肤、鳍条和鳞片中的分布和表达情况。结果显示,spr cDNA全长879 bp,包含132 bp和134 bp的5′和3′非编码区,开放阅读框510 bp,编码170个氨基酸残基。氨基酸序列比对和系统进化树分析显示锦鲤SPR具有保守的adh_short_C2结构域,与金鱼相似性高达97.7%。spr在各组织中均有表达,其中皮肤的表达量最高。spr在锦鲤个体发育的四个阶段表现为先降后升。纯红、纯白及红白三种体色锦鲤皮肤、鳞片和鳍条中spr mRNA和蛋白表达水平基本一致,在纯红锦鲤皮肤中表达量最高,红白锦鲤白色皮肤、鳞片和鳍条的表达量最低。SPR组织定位分析显示,红色锦鲤和白色锦鲤皮肤中均检测到阳性信号,其中红色皮肤阳性信号强度高于白色皮肤。以上结果表明,spr基因可能与锦鲤红/黄色素细胞的分化和形成具有一定相关性,参与了锦鲤体色的形成。
英文摘要:
      the origin of teleost skin color is from chromatophores. Different chromatophores synthesize distinct pigments, including melanin, carotenoid, pteridine and purine. Sepiapterin is a yellow pteridines, which could be transformed into tetrahydrobiopterin (BH4) and yellow/red pteridines pigments together with sepiapterin reductase (SPR), dihydrofolate reductase (DHFR) and other enzymes. SPR is the last enzyme in the process of de novo BH4 synthesis. In order to explore the function of spr in koi carp color formation, this study amplified the whole cDNA of spr and analyzed the spatio-temporal profile. Furthermore, we also detected the expression and distribution of SPR in the skin, fins and scales of koi carp with different colors by western blot and immunochemistry methods. The results showed that the size of spr cDNA was 879 bp, including 132 bp and 134 bp 5′ and 3′ untranslated regions, and a 510 bp open reading frame encoding 170 amino acids. Sequences alignment and phylogenetic analysis revealed the spr gene of koi carp contained a adh_short_C2 conserved domain and had 97.65% similarity with gold fish. spr was expressed in every tissues, especially highest expressed in skin. In the four ontogenetic stages, the expressive level of spr firstly decreased then rose. The expression level of spr mRNA and protein in skins, fins and scales displayed the same condition among three colors (whole red koi carp, whole white koi carp and kohaku koi carp). The highest expressive level was detected in whole red koi carp and rarely in white skin, fins and scales of kohaku koi carp. The immunohistochemical positive signals were detected in both skins of whole red and whole white koi carp, and intensively exhibited in red skin comparing with white skin. All the results might infer the spr gene has relationship with the xanthophores/erythrophores differentiation and formation, involving in koi carp color formation.
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