卵形鲳鲹Myostatin基因克隆及其在胚胎发育中的表达分析

Molecular Cloning of the Myostatin Gene and Its Expression During Embryo Development of Trachinotus ovatus

为研究肌生长抑制素(myostatin,MSTN)在卵形鲳鲹胚胎发育过程中的表达,对卵形鲳鲹MSTN基因CDS区进行克隆与分析,并对其14个胚胎发育阶段的表达量进行研究。结果显示:克隆获得MSTN基因1322 bp序列。该序列编码376个氨基酸,相对分子质量为42694.85 u,理论等电点pI为5.52,为带负电的蛋白。MSTN蛋白质不存在跨膜结构域,亚细胞定位主要于细胞外部,可能属于分泌蛋白质,易于表达和纯化。MSTN蛋白质无信号肽序列,预测含有17个磷酸化位点。对24种鱼类MSTN基因序列的同源分析发现:卵形鲳鲹MSTN与布氏鲳鲹同源性最高,与黄尾鰤同源性次之;3种鱼类均属鲹科Carangidae,而鲹科鱼类与锯盖鱼科Centropomidae鱼类遗传距离最小,具有较近的亲缘关系。MSTN基因在卵形鲳鲹从受精卵到晶体出现期的13个阶段均表达量极低或不表达,组间差异不显著(P>0.05),而在初孵仔鱼期表达量迅速升高,极显著高于前13个时期(P<0.01)。MSTN基因可能在卵形鲳鲹孵化后发挥重要作用,而在孵化前作用微弱。

Myostatin(MSTN)is a negative regulator of skeletal muscle growth,and much interest has been given to its role in animal development in recent years.In this study,MSTN gene in Trachinotus ovatus was cloned and characterized,and the expression patterns of 14 embryonic development stages of T.ovatus were studied.The results showed that 1322 bp of MSTN gene was cloned,encoding a 376 amino acid polypeptide.The relative molecular weight was 42694.85 u,and the theoretical isoelectric point pI was 5.52,which was a negatively charged protein.MSTN protein had no transmembrane domain and subcellular localization was mainly outside the cell,which may be secretory protein and easy to express and purify.MSTN protein had no signal peptide sequence,and 17 phosphorylation sites were predicted.A phylogenetic analysis of 24 species of fish revealed that the MSTN of T.ovatus had the highest homology with T.blochii,followed by that of S.lalandi dorsalis,all of which belonged to Carangidae,while that of Carangidae and centropomidae had the lowest genetic distance and close relationship.MSTN gene was very low or not expressed in all 13 stages of T.ovatus from Fertilized egg to Formation of eye lens stage,with no significant difference between the groups(P>0.05).However,the expression level of MSTN gene increased rapidly in the hatching stage,which was significantly higher than that in the previous 13 stages(P<0.01).The MSTN gene may play an important role after the T.ovatus hatched,but it is weak before hatching.