草鱼生肌因子5基因的克隆及其组织表达谱分析

Cloning and Expression Profiling of Myf-5 Gene of Grass Carp

目的：获得草鱼生肌因子5（Myf-5）基因序列,分析其在草鱼不同组织和不同发育阶段中的表达规律。方法：根据鲤鱼Myf-5基因序列设计引物,用草鱼肌肉组织总RNA,经RT-PCR扩增其Myf-5基因序列;利用半定量RT-PCR分析草鱼Myf-5基因在草鱼不同组织和不同发育阶段的mRNA表达特性。结果：获得了草鱼Myf-5基因开放读框序列723 bp,GenBank登录号为GU290227;该基因编码由240个氨基酸残基组成的蛋白,具有MyoD家族基因的典型性碱性螺旋-环-螺旋（bHLH）结构,其氨基酸序列与斑马鱼、鲤鱼、虹鳟、大西洋鲑等的同源性较高（74%-97%）,与哺乳动物和禽类如人、小鼠、大鼠、猪、牛和鸡的同源性较低（56%-60%）;在草鱼红肌、白肌、肝胰脏、肾脏、脑和肠中均检测到Myf-5基因的表达,红肌、白肌和脑组织中Myf-5基因mRNA的表达量显著高于其他组织（P〈0.05）;草鱼Myf-5基因的表达随着其生长发育呈下降趋势,在较大规格试验鱼（500 g）中的表达显著低于其他2种规格（50-60 g、120-130 g）的试验鱼（P〈0.05）。结论：获得了草鱼Myf-5基因序列,其在红肌、白肌和脑组织中的表达量显著高于其他组织,并随生长发育呈下降趋势,为研究Myf-5在草鱼肌肉发育过程中的作用提供了基础资料。

Objective： To obtain the myogenic factor 5（Myf-5） gene sequence of grass carp and analyze it＇s expression pattern in the different muscle tissue and developmental stage. Methods： PCR primers were designed according to Myf-5 gene sequence of Cyprinus carpio. Using the total RNA of grass carp muscle tissue and RT-PCR method, we amplified Myf-5 gene sequence; we also analyzed the mRNA expression character of Myf-5 gene, using semi-quantitative RT-PCR, in different tissue and developmental stage of grass carp. Results： A 723 bp ORF（GenBank accession No. GU290227） of Myf-5 gene was obtained with RT-PCR. The eDNA, encodes 240 amino acids peptides, with typical MyoD gene family structure trait of basic helix-loop-helix（b-HLH）. Compared with zebrafish, common carp, rainbow trout and atlantic salmon, the homology of amino acid sequences were higher （74%-97%）, but lower in some mammals and poultry（56%-60%）. The Myf-5 gene expression was observed in red muscle, white muscle, hepatopancreas, kidney, brain and intestines, and the expression level in red muscle, white muscle and brain was significantly higher than that of other tissues studied（P〈0.05）. The expression of Myf-5 gene decreased in grass carp with growth, showing weaker in larger size fish（500 g） than in other two smaller size groups（50-60 g, 120~130 g）（P〈0.05）. Conclusion： We successfully obtained the Myf-5 gene se- quence of grass carp, and it＇s expression level in red muscle, white muscle and brain was significantly higher than that of other tissues and expression level decreased in grass carp with growth. We offered a initial value for the role of Myf-5 on the muscle tissue development of grass carp.