文蛤Smad1/5基因克隆、时空表达及生长相关SNP位点筛查

CLONING, SPATIOTEMPORAL EXPRESSION AND SNPS DETECTION OF SMAD1/5 GENE IN HARD CLAM MERETRIX MERETRIX

  • 摘要: 为探讨Smad1/5基因在贝类生长发育中的调控作用, 利用RACE技术克隆获得文蛤Smad1/5(Mm-Smad1/5)基因的cDNA全长序列, 对其生物信息学、不同组织和不同发育时期时空表达特征进行分析, 并利用直接测序法分析了外显子区域SNP位点与生长性状的相关性。结果表明: Mm-Smad1/5的cDNA全长序列为1832 bp, 开放阅读框1380 bp, 编码459个氨基酸; 氨基酸多序列比对显示, Mm-Smad1/5蛋白与太平洋牡蛎Smad5、大西洋舟螺Smad1的一致性分别为83.7%和80.2%, 与人、鸡、非洲爪蟾等脊椎动物Smad1、Smad5氨基酸序列的一致性达到70.5%以上, 说明该基因具有较高的保守性; 结构域预测发现, Mm-Smad1/5含有Smads蛋白家族特有MH1、MH2两个高度保守结构域。荧光定量PCR(qRT-PCR)结果表明, Mm-Smad1/5基因在成体6个组织均有表达, 尤其在斧足、外套膜中表达量显著高于其他组织(P<0.05);Mm-Smad1/5基因在各发育时期广泛表达, 从原肠胚期开始大量表达, 一直持续到壳顶幼虫期, 而从眼点幼虫大量下降, 稚贝时期又有所上升。Mm-Smad1/5基因外显子区域SNP位点相关分析表明, 共发现了9个SNP位点, 其中936 G>T位点与文蛤的生长性状显著相关(P<0.05)。Mm-Smad1/5基因在文蛤生长发育中发挥重要调控作用, 可作为高产良种选育的候选基因, 而生长关联SNP位点分析将为文蛤分子标记辅助育种研究奠定重要基础。

     

    Abstract: To study the biological function of Smad1/5 in the growth and development of molluscs, the full-length cDNA of Smad1/5 in Meretrix meretrix (Mm-Smad1/5) was cloned via SMART RACE techniques. The entire cDNA of Mm-Smad1/5 was 1832 bp long, containing a 1380-bp open reading flame (ORF) that encoded a 459-amino acid protein. According to the amino acid sequence alignment, Mm-Smad1/5 shared 83.7% identity with Smad5 of C. gigas, 80.2% with Smad1 of P. fucata, and more than 70.5% identity with vertebrates. Protein functional domain predictions revealed that Mm-Smad1/5 contained both of the highly conserved functional domains MH1 and MH2. Together, these data indicate that Smad1/5 is highly conserved across evolution. The qRT-PCR results showed that Mm-Smad1/5 were expressed in all six tissues of adult clams and were expressed significantly more in the foot and mantle than other tissues (P<0.05). Furthermore, theMm-Smad1/5 gene was widely expressed in 10 developmental stages, it was first highly expressed in the gastrulae until the eyebot larva, largely decreased in the eyebot larva, and slightly increased in juvenile clams. Additionally, we identified 9 potential SNPs in the exons of Mm-Smad1/5 by DNA sequencing, of which the 936 G>T locus was significantly with the growth traits ofM. meretrix (P<0.05). The study indicated thatMm-Smad1/5 gene could be used as the growth candidate function gene, and its SNPs provided useful information for the reliable molecular markers for M. meretrix breeding.

     

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