瘤背石磺肌肉生长抑制素基因的克隆及组织表达分析

Cloning and tissue expression analysis of the MSTN gene in Onchidium struma

为探究石磺由海洋向陆地进化过程中肌肉生长和发育的分子机制,实验以石磺科贝类转录组数据为基础,采用RACE方法从瘤背石磺肌肉中首次克隆到MSTN cDNA的全长并做了相应的组织表达分析。结果显示,瘤背石磺MSTN基因cDNA全长2667 bp,包括1650 bp的开放阅读框,374 bp的5′端非翻译区,643 bp的3'端非翻译区,共编码549个氨基酸。预测该基因编码的蛋白质原子总量为8776,分子式为C2774H4331N783O862S26,分子质量约为63.27 ku,理论等电点为6.02,信号肽预测结果显示,N端具有21个氨基酸长度的信号肽。瘤背石磺MSTN具有MSTN的共同特征,包括蛋白酶水解位点RSRR和C端多肽生物活性区以及9个保守的半胱氨酸残基。通过进化树分析,瘤背石磺MSTN与加州海兔MSTN的亲缘关系最近。RT-PCR结果显示,MSTN基因在各个组织中均有表达;含肌纤维组织中的表达量低于内脏器官的表达量,在肝胰腺中的表达量最高,腹足表达量最低。MSTN基因一级结构具有很高的保守性,说明该基因在进化上的限制性和功能的重要性;同时该基因在石磺非肌肉组织中表达,表明该基因不仅有抑制肌肉生长的作用,还参与其他生命活动的调节。

In this study, we used rapid amplification cDNA ends （RACE） methods to obtain the full-length cDNA of Myostatin gene in Onchidium struma. The bioinformatic analysis and expression pattern of MSTN mRNA in different tissues detected by Real-time fluorescent quantitative PCR （qRT-PCR） were performed. The full length of MSTN cDNA sequence consists of 2667 base pairs （bp）, comprising a 374 bp 5＇ untranslated region （UTR）, a 643 bp 3＇UTR, and a1650 bp open reading frame （ORF） which encodes 549 amino acids. The MSTN protein was predicted to contain 8776 atoms and its formula is C2774H4331N7830862S26, with a calculated relative molecular weight of 63.27 kD and pI of 6.02. The result of signal peptide prediction shows that the N-terminal has a signal peptide of 21 amino acids length. The common features were found in MSTN of O. struma, including a conservative hydrolytic site （RSRR） and 9 cysteine residues. The phylogeny analysis shows that the relationship between O. struma and Aplysia californica was the closest. According to RT-PCR results, MSTN gene was expressed in various tissues with the highest expression in the hepatopancreas and lowest in foot; expression in muscle is lower than that in internal organs, and expression in muscle of abdominal skin is higher than other parts. Our results suggest that MSTN gene may play an important role in muscle growth and development in O. strurna, and may provide useful information for further studies on evolution of marine invertebrates from sea to wetland.