摘要:
Myostatin is an important member of the transforming growth factor (TGF) family that functions to reg-ulate muscle development and growth in animals, The purpose of this study was to characterize and predict func-tion of the myostatin gene of Haliotis diversicolor supertexta which is an important aquaculture shellfish. In this study, the myostatin (Hs-MSTN) cDNA of H. diversicolor supertexta were cloned and characterized by rapid amp-lification cDNA ends (RACE) methods. The full length of Hs-MSTN cDNA sequence consists of 3 755 bp contain-ing a 5′ untranslated region (UTR) of 324 bp, a 3′ UTR of 1 985 bp, and an open reading frame of 1 446 bp encod-ing a protein with 481 amino acid residues, with a calculated molecular mass of 54.96 ku, and the theoretical iso-electric point of 9.41, The structure of Hd-MSTN included a putative signal peptide (1-17 aa), a TGF-β propeptide domain (157-367 aa) and a conserved TGF-β domain (379-481 aa). Multiple sequence alignment results revealed conservation of the RRPR proteolytic site and nine conserved cysteines of the Hs-MSTN with MSTN from other animals, and two propeptide proteolytic sites RQRR (120-124 aa) and RYRR (235-239 aa) were found. Phylogen-etic analysis showed that the Hs-MSTN gene was clustered in the same subgroup with the H. rufescens, Quantitat-ive real-time PCR detection results indicated that the Hs-MSTN genes were expressed widely in adductor muscle, mantle, gonad, liver, gill, foot and the highest expression level was observed in the adductor muscle, mantle, foot, and Hs-MSTN transcript was widely detected in early developmental stages: unfertilized egg, fertilized egg, 4-cell embryos, 8-cell embryos, gastrulae, larvae, juvenile stage, and the higher in fertilized egg, in 4-cell embryos and 8-cell embryos and the expression level increases in gastrulae, larvae and decreases in juvenile stage. Our results in-dicate that MSTN is involved in muscle growth regulation of H. diversicolor supertexta.%肌肉生长抑制素(MSTN)是转化生长因子β超家族(TGF-β)中参与动物肌肉生长的重要调控因子.为了解MSTN基因在九孔鲍中的功能,本研究采用cDNA末端快速扩增(RACE)技术从九孔鲍右侧壳肌中获得了MSTN基因cDNA全长,并运用实时荧光定量PCR (qRT-PCR)技术检测了MSTN在各组织和发育时期的表达水平.结果显示,九孔鲍cDNA全长3 755 bp,其中5′非编码区(5′UTR)324 bp,3′非编码区(3′UTR)1 985 bp,开放阅读框(ORF) 1 446 bp,编码481个氨基酸,分子质量为54.96 ku,理论等电点pI为9.41;具有N端信号肽(1~17 aa)、 TGF-β前肽区域(157~367 aa)和成熟肽区域(379~481 aa),以及蛋白酶水解位点RRPR (364~368 aa)和C端生物活性区9个保守的半胱氨酸残基,符合TGF-β超家族蛋白典型结构特征,且预测到2个新的蛋白酶水解位点RQRR (120~124 aa)、RYRR (235~239 aa).系统进化树结果显示,九孔鲍MSTN基因和红鲍MSTN基因聚为一支.qRT-PCR结果表明,九孔鲍MSTN基因在检测的6个组织中均表达,且在足、右侧壳肌、外套膜中高表达,在鳃、性腺、肝脏中低表达;在检测的7个发育时期均表达且在受精卵、原肠胚、稚鲍时期高表达,在卵、4细胞期、8细胞期、幼鲍时期表达量较低.研究表明MSTN基因可能在九孔鲍肌肉生长中具有重要作用.