九孔鲍MSTN基因cDNA克隆及表达

Molecular cloning and expression analysis of the MSTN cDNA in Haliotis diversicolor supertexta

摘要:

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。

关键词:九孔鲍;肌肉生长抑制素;基因克隆;基因表达

Abstract:

Myostatin is an important member of the transforming growth factor (TGF) family that functions to regulate muscle development and growth in animals, The purpose of this study was to characterize and predict function 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 amplification cDNA ends (RACE) methods. The full length of Hs-MSTN cDNA sequence consists of 3 755 bp containing a 5' untranslated region (UTR) of 324 bp, a 3' UTR of 1 985 bp, and an open reading frame of 1 446 bp encoding a protein with 481 amino acid residues, with a calculated molecular mass of 54.96 ku, and the theoretical isoelectric 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. Phylogenetic analysis showed that the Hs-MSTN gene was clustered in the same subgroup with the H. rufescens, Quantitative 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 indicate that MSTN is involved in muscle growth regulation of H. diversicolor supertexta.

Key words:Haliotis diversicolor supertexta;myostatin;gene cloning;gene expression