鞍带石斑鱼MRFs基因cDNA克隆及组织表达分析

Gene cloning and expression analysis of MRFs gene cDNA in the tissues of giant grouper Epinephelus lanceolatus

摘要

生肌调节因子（MRFs）在动物体骨骼肌生长发育过程中发挥着重要作用。本研究通过 RT-PCR 和 RACE 技术克隆得到鞍带石斑鱼 5 种生肌调节因子（MRFs）cDNA 全长序列，包括 MyoD1（1 962 bp）、MyoD2（1 466 bp）、MyoG（926 bp）、MRF4（1 052 bp）和 Myf5（1 133 bp），分别编码 297、270、251、238 和 242 个氨基酸。通过氨基酸序列比对分析发现，5 种 MRFs 均含有由 60 个氨基酸组成的碱性螺旋−环−螺旋（bHLH）保守结构域。进化树分析显示，MRFs 分为两支，其中 MyoD（MyoD1 和 MyoD2）与 Myf5 聚为一支，MRF4 与 MyoG 聚为另一支，且各基因均与鲈形目鱼类聚为一支。实时荧光定量 PCR 结果显示，5 种 MRFs 在骨骼肌中表达量较高，而在肝脏、心脏和肠道等组织中表达较低。该研究为揭示 MRFs 在鞍带石斑鱼骨骼肌生长发育中的作用机制奠定了基础。

Abstract

Myogenic regulatory factors (MRFs) are critical regulators of skeletal muscle growth and development in animals. The full-length cDNA sequences of five MRFs from the giant grouper (Epinephelus lanceolatus) were cloned using RT-PCR and RAC, including MyoD1 (1,962 bp), MyoD2 (1,466 bp), MyoG (926 bp), MRF4 (1,052 bp), and Myf5 (1,133 bp), encoding 297, 270, 251, 238, and 242 amino acids, respectively. Amino acid sequence alignment revealed that all five MRFs contain a conserved basic helix-loop-helix (bHLH) domain consisting of 60 amino acids. Phylogenetic analysis demonstrated that the MRF genes are clustered into two distinct branches: MyoD (MyoD1 and MyoD2) groups with Myf5 in one clade, while MRF4 and MyoG form the other. All genes showed closest evolutionary relationships with Perciformes homologs. Real-time quantitative PCR analysis revealed predominant expression of these MRFs in skeletal muscle, with significantly lower expression levels in the liver, heart, and intestine. These findings provide foundation for elucidating the regulatory mechanisms of MRFs in skeletal muscle development of the giant grouper.

关键词

鞍带石斑鱼 / 生肌调节因子 / cDNA 克隆 / 基因表达

Key words

Epinephelus lanceolatus / myogenic regulatory factors / cDNA cloning / gene expression

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基金资助

国家自然科学基金地区科学基金项目(32260919)