九孔鲍BMP-2基因cDNA克隆及表达分析

Molecular cloning and expression analysis of BMP-2 cDNA in Haliotis diversicolor supertexta

为探究BMP-2基因在九孔鲍(Haliotisdiversicolorsupertexta)中的功能,采用cDNA末端快速扩增(RACE)技术从九孔鲍外套膜中获得了BMP-2基因cDNA全长,并用实时荧光定量PCR(qRT-PCR)检测了BMP-2基因在各组织和发育时期的表达水平。结果表明:九孔鲍BMP-2基因cDNA全长2572bp,其中5′非编码区(5′UTR)123bp, 3′非编码区(3′UTR)1150bp,开放阅读框(ORF)为1299 bp,编码432个氨基酸,其分子质量为48.59ku,理论等电点(pI)为9.84;具有N端信号肽(1-39 aa)、TGF-β前肽区域(63-294 aa)和TGF-β成熟肽区域(331-432 aa),以及蛋白酶水解位点RLRR (272-275 aa)和7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。系统进化树结果显示九孔鲍BMP-2基因和耳鲍聚为一支。qRT-PCR结果表明BMP-2基因在九孔鲍的6个组织中均有表达,在足、右侧壳肌、外套膜及肝脏中显著高表达;在检测的7个发育时期均表达,其中在受精卵、4细胞、8细胞、原肠胚、稚鲍时期表达量显著高于卵、幼鲍时期。研究表明BMP-2基因可能在九孔鲍贝壳形成中具有重要作用。

This study was conducted to characterize and predict the function of the BMP-2 gene of Haliotis diversicolor supertexta, an important aquaculture shellfish. The bone morphogenetic protein 2 (Hs-BMP-2) cDNA of H. diversicolor supertexta was cloned and characterized by the rapid amplification of cDNA ends (RACE) method. The full-length Hs-BMP-2 cDNA sequence consisted of 2572 bp containing a 5′ untranslated region (UTR) of 123 bp, a 3′ UTR of 1150 bp, and an open reading frame consisting of 1299 bp encoding a protein with 432 amino acid residues, whose calculated molecular mass was 48.59 ku and the theoretical isoelectric point was 9.84. The structure of Hs-BMP-2 included a putative signal peptide (1–39 aa), a TGF-β propeptide domain (63–294 aa), and a conserved TGF-β domain (331–432 aa). Multiple sequence alignment results revealed conservation of the RLRR (272–275 aa) proteolytic site and seven conserved cysteines of Hs-BMP-2 with BMP-2 from other animals. Phylogenetic analysis revealed that the Hs-BMP-2 gene was clustered in the same subgroup with H. asinina. Quantitative real-time PCR detection results indicated that the Hs-BMP-2 gene was widely expressed in the adductor muscle, mantle, gonad, digestive gland, gill, and foot, with the highest expression level in the adductor muscle, mantle, foot, and digestive gland. The Hs-BMP-2 transcript was widely detected in early developmental stages of the unfertilized egg, fertilized egg, 4-cell embryos, 8-cell embryos, gastrulae, larvae, and juvenile stage, with higher transcript levels being detected in the fertilized egg, 4-cell embryos, 8-cell embryos, larvae, and juvenile stage. These results indicate that BMP-2 is involved in shell growth regulation of H. diversicolor supertexta.