锦鲤microRNA-137系统进化、靶基因验证及表达分析
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Q786;S917.4

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国家自然科学基金(31402294);河南省科技攻关计划(农业领域)(192102110192)


Evolution, target genes certification and expression analysis of microRNA-137 in Japanese ornamental carp (Cyprinus carpio var. koi)
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    摘要:

    为了探究 microRNA-137(miR-137)与锦鲤体色形成的关系,实验对已报道的14种鱼类miR-137前体序列(precursor miR-137, pre-miR-137)进行比对,利用最大似然估计法(Maximum Likelihood Estimate, MLE)构建系统进化树,同时荧光定量PCR (quantitative real-time PCR, qRT-PCR)分析其在体色发生阶段和不同组织中的相对表达水平,并预测其靶基因,随后对靶基因进行GO和KEGG分析,最后通过荧光素酶实验验证miR-137-3p与靶基因的靶向关系,并分析miR-137-3p与靶基因mRNA的表达变化。结果显示,锦鲤与鲤的pre-miR-137序列相似度最高,且pre-miR-137序列在所有鱼类中具有较高的保守性。对miR-137靶基因进行GO和KEGG分析,多个靶基因富集在色素沉积、色素细胞分化等信号通路。定量结果显示,锦鲤出膜后11 d(day post hatching, dph) miR-137-3p表达量达到最高,随后显著降低;miR-137-3p在锦鲤不同组织中均有表达,在眼和肌肉中表达量较高,在皮肤和鳍条等色素细胞存在的组织中也有较高表达,且白色组织表达量显著高于红色组织。荧光素酶实验结果显示,miR-137-3p可结合在mitfa 3′-UTR上并抑制其表达,但对sprb并无显著抑制作用;miR-137-3p与mitfasprb在不同发育阶段存在显著负相关,但在组织中不存在相关性。本研究发现,miR-137在鱼类进化过程中高度保守,与锦鲤体色形成具有一定的相关性,且可靶向调控mitfa参与体色的形成,以上结果为进一步探讨 miR-137在锦鲤体色形成中的作用提供基础数据。

    Abstract:

    MicroRNAs (miRNAs) play an crucial role in post-transcriptional regulation of most transcripts in almost all animal species. Researchers have discovered that many miRNAs and a subset of their targets are conserved across deeply diverged phyla. miR-137 has been identified to function in melanogenesis in mammals. Considering the relation of miR-137 with melanin synthesis and conservation in species evolution, it might exert efforts in fish pigment synthesis. However, the function of miR-137 in fish color formation is still unclear. The Japanese ornamental carp (C. carpio var. koi) is famous for various colors and widely cultured all over the world. Due to its fast establishment of various colors under cultivation, C. carpio var. koi is a distinctive specie for genetics research in pigmentation. In order to explore the role of miR-137 in the color formation of C. carpio var. koi, the precursor sequences of miR-137 in 14 fish species were aligned and phylogenetic tree was constru-cted by Maximum Likelihood Estimate. Besides, the expression levels of miR-137 in different developmental stages and tissues also detected. Then, the target genes of miR-137 were predicted and analyzed by GO and KEGG analysis. In addtion, the luciferase reporter was used to verify the availability of miR-137 with its targets, and the expression levels of miR-137 and target genes in different development stages and tissues were checked and the correlation analysis between them was also conducted. The results showed that the precursor sequence similarity of miR-137 between C. carpio var. koi and C. carpio was the highest, and miR-137 sequences exhibited high conservation among all fish species. A great number of predicted target genes were enriched in melanogenesis, pigment cell differentiation, pteridine-containing compound metabolic process and so on. Real time PCR results showed the expression of miR-137-3p Feached the peak on 11 dph and rapidly dicreased in the following stages. miR-137 was expressed in all kinds of tissues, especially significantly in eye and muscle. Besides, the skin and fins, pigment cell enriched tissues, also had a higher expression and the expression of miR-137 in white tissues was higher than the red. The luciferase reporter analysis certified that miR-137 bound with the mitfa 3′-UTR to inhibit its expression but did not present significant inhibitory effect on sprb, and miR-137 was negatively correlated with the expression of mitfa and sprb in different developmental stages, but not in tissues. All the above results confirmed miR-137 was highly conserved during fish species evolution and displayed correlation with the koi color formation, in which miR-137 might regulate mitfa involved in color formation. This study provides basic research data for further exploring the role of miR-137 in C. carpio var. koi color formation.

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李俊茹,马晓,吴利敏,刘慧芬,石西,宋红梅,田雪,李学军.锦鲤microRNA-137系统进化、靶基因验证及表达分析[J].水产学报,2021,45(6):831~845

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  • 收稿日期:2020-10-10
  • 最后修改日期:2020-12-07
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  • 在线发布日期: 2021-06-16
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