生物学杂志

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蓝点马鲛mstn克隆及表达分析

  

  1. 宁波大学海洋学院
  • 出版日期:2015-12-18 发布日期:2015-12-18
  • 通讯作者: 薛良义,博士,教授,研究方向:分子生物学,E-mail:xueliangyi@nbu.edu.cn。

Cloning and expression analysis of mstn in Scomberomorus niphonius

  1. School of Marine Science, Ningbo University
  • Online:2015-12-18 Published:2015-12-18

摘要: 利用同源克隆和cDNA末端快速扩增(RACE)技术克隆了蓝点马鲛肌肉生长抑制素基因(mstn),并分析了mstn的结构特征和亲缘关系。克隆的蓝点马鲛mstn共3870bp,包括3个外显子1131bp和2个内含子1201bp。外显子Ⅰ379bp,内含子Ⅰ374bp,外显子Ⅱ371bp,内含子Ⅱ827bp,外显子Ⅲ381bp,5’UTR 108bp,3’UTR 1430bp。3’UTR含有加尾信号AATAAA。mstn共编码376个氨基酸,包括信号肽(1aa~22aa)、TGF-β前肽区域(37aa~257aa)和TGF-β功能域(282aa~376aa)。在内含子Ⅱ和3’UTR发现微卫星序列,分别为(CA)5和(CA)10TG(CA)6CT(CA)10。蓝点马鲛MSTN具有脊椎动物MSTN的共同特征,含一个蛋白酶水解位点RARR和9个保守的半胱氨酸残基。脊椎动物MSTN氨基酸序列的亲缘关系分析发现,蓝点马鲛与鳜鱼亲缘关系最近,与其他鲈形总目鱼类聚为一簇。以β-actin为内参基因,qPCR分析表明,蓝点马鲛mstn在二龄鱼不同组织表达情况不同,在鳃、肝和肾中表达较高,而在肌肉、肠、胃、脾和性腺中表达量较低。这些结果可为蓝点马鲛肌肉生长和发育的分子机制研究提供信息。

Abstract: Scomberomorus niphonius is widely distributed in China’s coastal area. It is a kind of migratory species and has been a very important part in China’s fishery catches till now. mstn has attracted the attention of large numbers of researchers since its important effect on muscle growth. Homology cloning and rapid amplification of cDNA ends (RACE) were employed to clone mstn of S. niphonius, and its structural characteristics and phylogenetic relationship were also analyzed in this paper. The length of cloned mstn was 3870bp, including 1131bp of 3 exons and 1201bp of 2 introns. ExonⅠ, intronⅠ, exonⅡ, intronⅡ and exonⅢ were 379bp, 374bp, 371bp, 827bp, and 381bp, respectively. 5’UTR and 3’UTR were 108bp and 1430bp, respectively. A polyA signal AATAAA was found in 3’UTR. The whole sequences encoded 376 amino acids, containing a putative signal peptide (1aa~22aa), a TGF-β propeptide domain (37aa~257aa) and a TGF-β functional domain (282aa~376aa). Two microsatellites were found, (CA)5 in intronⅡ and (CA)10TG(CA)6CT(CA)10 in 3’UTR. The similar microsatellites were also found in other fishes, which suggested that these regions might have some special functions. The common domains of vertebrate MSTNs were found in S. niphonius MSTN, including a conservative hydrolytic site (RARR) and 9 cysteine residues. The phylogeny analysis showed that the relationship between S. niphonius and Siniperca chuatsi was the closest, and S. niphonius grouped into the same branch with other Percomorpha fishes. mstn expressions in different tissues were examined by qPCR, and β-actin was employed as housekeeping gene. qPCR results showed that mstn transcriptional level varied among different tissues in two years old S. niphonius, with a high expression in gill, liver and kidney, and a low expression in muscle, intestine, stomach, spleen and gonad. These results provided useful information for the study of the molecular mechanism of S. niphonius muscle growth and development.