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吴深基,赵璐(共同第一作者),黄进强,李永娟,张倩,潘玉财,王晓谰,刘哲.2021.虹鳟mc1r基因的克隆、序列分析及在 不同发育阶段和组织的表达分析.动物学杂志,56(5):746-755.
虹鳟mc1r基因的克隆、序列分析及在 不同发育阶段和组织的表达分析
Cloning, Sequencing and Expression Analysis of mc1r Gene in Different Developmental Stages and Tissues of Rainbow Trout Oncorhynchus mykiss
投稿时间:2021-01-05  修订日期:2021-08-23
DOI:10.13859/j.cjz.202105011
中文关键词:  虹鳟  体色  黑素皮质素1受体基因(mc1r)  基因克隆  表达分析
英文关键词:Rainbow Trout (Oncorhynchus mykiss)  Body color  Melanocortin 1 receptor gene (mc1r)  Cloning  Expression analysis
基金项目:国家自然科学基金项目(No. 31760755)
作者单位E-mail
吴深基 甘肃农业大学动物科学技术学院 兰州 730070 1753548729@qq.com 
赵璐(共同第一作者) 甘肃农业大学动物科学技术学院 兰州 730070 1283267706@qq.com 
黄进强 甘肃农业大学动物科学技术学院 兰州 730070 huangjinq@163.com 
李永娟 甘肃农业大学理学院 兰州 730070 flyinglyj@126.com 
张倩 甘肃农业大学动物科学技术学院 兰州 730070 983974150@qq.com 
潘玉财 甘肃农业大学动物科学技术学院 兰州 730070 1140460456@qq.com 
王晓谰 甘肃农业大学动物科学技术学院 兰州 730070 1807943648@qq.com 
刘哲 甘肃农业大学动物科学技术学院 兰州 730070  
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中文摘要:
      黑素皮质素1受体基因(mc1r)是控制动物色素合成的重要基因,为探讨mc1r基因与虹鳟(Oncorhynchus mykiss)体色变异的关系,本研究利用cDNA末端快速扩增(RACE)技术获得虹鳟mc1r基因的cDNA全长序列,并对其编码的蛋白进行了生物信息学分析,同时利用实时荧光定量PCR(qRT-PCR)分析该基因在野生型虹鳟(虹鳟)和黄色突变型虹鳟(金鳟)体色发生不同时期(从受精期至12月龄)及成鱼背部皮肤、腹部皮肤、背部肌肉、腹部肌肉、眼、脑、鳃、中肾、头肾、肠、肝、脾和心13种组织中的表达差异。结果显示,mc1r基因序列全长为4 518 bp,开放阅读框1 017 bp,编码338个氨基酸。氨基酸序列分析发现,虹鳟Mc1r蛋白具有7TM_GPCR_Srsx结构域。通过氨基酸序列同源比对与系统进化分析表明,Mc1r蛋白序列在鱼类间具有较高的保守性。qRT-PCR结果表明,mc1r基因在虹鳟与金鳟的受精期就开始表达,且在受精期至桑葚期胚胎的表达量高于胚胎后期;mc1r基因在虹鳟与金鳟相同时期表达比较结果显示,该基因在受精期、4细胞期、16细胞期、囊胚期、原肠期、神经期、体节期、1日龄、3日龄、7日龄胚胎或个体以及1月龄、2月龄、3月龄和6月龄背部皮肤中的表达均差异显著(P < 0.05);mc1r基因在12月龄虹鳟和金鳟的13种组织中均有表达,其中,该基因在虹鳟与金鳟的背部皮肤、腹部皮肤和脑中的表达量较高,显著高于其他组织(P < 0.05),且虹鳟背部皮肤中该基因的表达量高于金鳟背部皮肤(P > 0.05)。以上结果表明,mc1r基因可能与虹鳟体色变异密切相关。本研究可为后期进一步深入阐明虹鳟体色变异的分子机制提供基础资料。
英文摘要:
      Melanocortin 1 receptor gene (mc1r) is an important gene that controls pigment synthesis in animals. To explore the relationship between mc1r gene expression level and body color variation in Rainbow Trout (Oncorhynchus mykiss), the present work obtained the full-length cDNA sequence of the gene by rapid-amplification of cDNA ends (RACE) technology, and the encoded protein was analyzed by bioinformatics. Simultaneously, quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of mc1r gene in different developmental stages (from fertilization stage to 12 months post hatch) and tissues (dorsal skin, ventral skin, dorsal muscle, ventral muscle, eye, brain, gill, midkidney, headkidney, intestine, liver, spleen and heart) of wild-type and yellow mutant Rainbow Trouts, respectively. The results showed that mc1r gene was 4 518 bp, containing 1 017 bp open reading frame (ORF) (encoding 338 amino acid residues), 532 bp 5? untranslated region (UTR), and 2 969 bp 3? UTR. Sequence analysis revealed that Mc1r protein of Rainbow Trout had the 7TM_GPCR_Srsx structural domain, and sequence analysis of amino acids and phylogenetic tree analysis indicated that conservation of Mc1r was higher in fish than that in other vertebrates (Table 2 and Fig. 2). qRT-PCR results showed that mc1r gene began to express at the fertilized egg stage of wild-type and yellow mutant Rainbow Trouts, and the expression in the early embryonic stages (from fertilized egg stage to multi-cell stage) was higher than that in the late embryonic stages (Fig. 3). Moreover, there were significant differences (P < 0.05) in the expression level of mc1r gene at the same stages between wild-type and yellow mutant Rainbow Trouts, including fertilized egg stage, 4-cell, 16-cell, blastula, gastrula, neurula, somite stage, 1 day post hatch, 3 days post hatch, 7 days post hatch, 1 month post hatch, 2 months post hatch, 3 months post hatch and 6 months post hatch (Fig. 3). mc1r gene also widely expressed in various tissues in 12 months post hatch wild-type and yellow mutant Rainbow Trouts, with the higher expression levels in dorsal skin, ventral skin and brain, when compared to other tissues (P < 0.05) (Fig. 4). Moreover, the expression level of mc1r gene in dorsal skin of wild-type was higher than that of yellow mutant Rainbow Trout (P > 0.05) (Fig. 4). In conclusion, the expression level of mc1r gene might be closely related to body color variation of Rainbow Trout. The results of this study provided valuable data for later studies on the molecular mechanism of Rainbow Trout body color variation.
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