淇河鲫肌肉生长抑制素基因的克隆与表达

Molecular cloning and expression analysis of myostatin gene in Carassius auratus in Qihe River

肌肉生长抑制素(MSTN)是动物肌肉发育和生长过程中的负调控因子。为了明确淇河鲫MSTN基因序列及其在肌肉生长发育中的调控功能,本研究利用RACE方法克隆淇河鲫MSTN全长c DNA序列,q RT-PCR分析其在不同组织和胚胎发育阶段的表达情况。结果显示,淇河鲫MSTN c DNA序列全长2094 bp(no.KC851952.1),包含86 bp 5′非编码区、880 bp 3′非编码区和1128 bp开放阅读框,编码375个氨基酸残基,前22个氨基酸残基为信号肽,34~256位氨基酸残基为TGF-β前肽域,281~375是TGF-β结构域,具蛋白酶水解位点RIRR和C端生物活性区的9个保守半胱氨酸残基。蛋白同源分析发现淇河鲫MSTN与鲤形目鱼类相似性较高,与哺乳动物和鸟类的相似性最低。系统进化分析显示,淇河鲫MSTN与鲫、鲤、鳡等鲤科鱼类亲缘关系较近。实时定量PCR分析表明,淇河鲫MSTN基因在各个组织中均有表达,脑中表达量最高,其次为肌肉和肝脏,肠道表达量最低。淇河鲫胚胎发育的各阶段MSTN基因均有表达,受精卵表达量最高,其次为囊胚期,神经胚期表达量最低。推测MSTN与淇河鲫肌肉发育生长具有一定的相关性,可能参与"双背鲫"特征的形成。

Myostatin（MSTN） is a negative regulatory factor in animal muscle development and growth. In order to identify the sequence information and the function of Carassius auratus MSTN in the process of muscle development,rapid amplification of c DNA ends（RACE） and real time PCR were used to clone the full length c DNA sequence and analyse the expression level of MSTN in embryonic development stages and different tissues.The results showed that the full length c DNA of MSTN was 2094 bp（no. KC851952.1）,including 86 bp at 5′-UTR,880 bp at 3′-UTR,and a 1128 bp open reading frame encoded a peptide of 375 amino acids. The putative peptide contained a 22 amino acids signal peptide,a TGF-β propeptide domain（34-256）,a TGF-β domain（281-375）,a conserved RIRR proteolytic processing site and 9 conserved cysteine residues in the C-terminal of the protein.Multiple sequence comparison indicated that the MSTN of Carassius auratus has high similarity with cypriniformes species,while has low similarity with mammalian and bird. The phylogenetic analysis also showed that the crucian carp MSTN has close relationship with cypriniformes species like Carassius carassius,Cyprinus carpio and Labeo rohita. Real time PCR results showed that MSTN m RNA was checked in eight different tissues of crucian carp,among which the maximum level was detected in brain,followed by muscle and liver,the minimum level was displayed in intestine. In addition,the MSTN was also confirmed to express at every stage of embryogenesis,in which the highest level was found at fertilized eggs,followed by blastula and the lowest level was detected at neurula. Thus,the MSTN gene was speculated to have relation with crucian carp muscle development and growth,and might play an important role in the ＂ double dorsal muscle＂ formation.