文章摘要
虹鳟pmela基因的克隆及其在不同发育阶段和组织中的表达分析
Cloning of pmela Gene in Rainbow Trout (Oncorhynchus mykiss) and its expression analysis in Different Developmental Stages and Tissues
投稿时间:2022-06-01  修订日期:2022-07-11
DOI:
中文关键词: 虹鳟  体色  前黑素小体蛋白a (pmela)基因  克隆  表达分析
英文关键词: Rainbow trout  Skin color  pmela gene  Cloning  Expression analysis
基金项目:国家自然科学基金项目
作者单位邮编
黄进强 甘肃农业大学动物科学技术学院 730070
王晓谰 甘肃农业大学动物科学技术学院 
李永娟 甘肃农业大学动物科学技术学院 730070
吴深基 甘肃农业大学动物科学技术学院 
赵璐 甘肃农业大学动物科学技术学院 
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中文摘要:
      前黑素小体蛋白a (premelanosome protein, pmela)基因是黑色素合成通路中的关键基因之一,对动物的体色有着重要影响。为探讨pmela基因在虹鳟(Oncorhynchus mykiss)体色变异中的作用,本研究利用cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术对pmela基因cDNA全长进行克隆并运用生物信息学方法分析该基因的序列特征,同时采用qRT-PCR比较pmela基因在野生型虹鳟(虹鳟)、黄色突变型虹鳟(金鳟)和杂交F1代受精期至孵化后3个月不同发育时期及成鱼不同组织中的相对表达量。研究获得pmela基因cDNA全长序列3 476 bp,包含2 532 bp开放性阅读框,编码843个氨基酸。序列分析发现Pmela属于疏水性蛋白且存在PKD功能结构域。同源性比对发现虹鳟Pemla氨基酸序列与红鲑(Oncorhynchus nerka)的同源性高达97.75%;进化分析结果显示虹鳟与红鲑的亲缘关系最近,与人类(Homo sapiens)和小鼠(Mus musculus)的亲缘关系最远。qRT-PCR结果显示,pmela基因在虹鳟与金鳟胚胎及出膜后各时期均有表达,在受精期至16细胞期中的表达为虹鳟高于金鳟,出膜后该基因在金鳟背部皮肤中的表达均高于虹鳟,且在4细胞期、16细胞期、原肠期、神经期、体节期、心跳期、1 dph (1 day post-hatching)、3 dph、5 dph、7 dph、10 dph、1 M (1 month post-hatching)和2 M时期中虹鳟与金鳟的表达存在显著差异。在各组织中,pmela基因在虹鳟与金鳟背部皮肤和眼睛中的表达显著高于其他组织。在杂交F1代中,pmela基因在不同发育时期和组织中的表达模式与双亲类似且在大部分相同时期和组织中的表达与双亲存在显著差异。上述结果表明,pmela基因的表达量高低与虹鳟体色具有一定的相关性,且可能参与其体色的形成过程。本研究结果为进一步研究pmela基因在虹鳟体色变异中的作用提供基础资料。
英文摘要:
      Premelanosome protein a (pmela) gene is one of the key genes in the melanin synthesis pathway, which has an important impact on skin color of animals. This study was conducted to explore the role of pmela gene in rainbow trout (Oncorhynchus mykiss) skin color variation. Firstly, the full-length cDNA sequence of pmel gene was obtained using rapid amplification of cDNA ends (RACE) technique, and the encoded protein was analyzed by bioinformatics. Secondly, qRT-PCR was performed to detect the relative expression of pmela gene in different developmental stages (from fertilized-stage to 3 months post-hatching (3 M)) and tissues of wild-type rainbow trout (WT), yellow mutant rainbow trout (YM) and their F1 generation. The results showed that the total length of pmela gene was 3 476 bp, including a 2 532 bp open reading frame encoding 843 amino acids. Bioinformatics analysis revealed that Pmela was a hydrophobic protein and had a PKD functional domain. Homology alignment showed that the similarity of Pmela amino acid sequence of rainbow trout with sockeye salmon (Oncorhynchus nerka) were 97.75%, and phylogenetic tree analysis indicated that rainbow trout had the closest relationship with sockeye salmon, and the furthest relationship with humans (Homo sapiens) and mice (Mus musculus). qRT-PCR results showed that pmela gene was expressed in all stages of WT and YM, and the expression level of the gene from fertilized-stage to 16-cell was higher in WT than in YM, and the opposite expression pattern was identified after hatching. Moreover, there were significant differences in the expression of pmela gene at the same stages between WT and YM, e. g., at the fertilized-stage, 4-cell, 16-cell, gastrula, neurula, somites, heartbeating, 1 dph (1 day post-hatching), 3 dph, 5 dph, 7 dph, 10 dph, 1 M and 2 M. In various tissues, pmela gene expression was significantly higher in the dorsal skin and eyes of WT and YM than in other tissues. In the hybrid F1 generation, the expression pattern of pmel gene was similar to those of both parents, and there was significant difference in expression at the same stages between F1 generation and parents in most of the same stages and tissues. The above results indicated that the expression level of pmela gene is correlated with the skin color of rainbow trout and may be involved in the formation of its skin color, which provide basic data for further studying the role of pmela gene in the variation of rainbow trout skin color.
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