黄条鰤MSTN基因的克隆及其在早期发育中的表达特征

Isolation of MSTN Gene cDNA and Its Expression Analysis During the Early Development of Yellowtail Kingfish Seriola aureovittata

为解析肌肉生长抑制素(Myostatin,MSTN)基因在黄条鰤(Seriola aureovittata)生长发育过程中作用机制。本研究采用cDNA末端快速扩增法,克隆了黄条鰤MSTN基因的全长cDNA序列,采用荧光定量PCR(RT-qPCR)技术对MSTN mRNA在黄条鰤不同组织、胚胎发育过程以及仔稚幼鱼发育过程中的差异表达特征进行了研究。序列分析结果显示,MSTN基因全长cDNA为1828 bp,开放阅读框为1131 bp,编码了376个氨基酸。RT-qPCR分析表明,在检测的脑、垂体、肝脏、肌肉、脾脏、肾脏、鳃、心脏、胃、肠、头肾组织中MSTN mRNA均有表达,其在肌肉中表达量最高(P<0.05)。MSTN mRNA在胚体下包2/3时期至孵化期表达量显著升高,且在孵化期达到峰值(P<0.05)。MSTN mRNA在孵化后3天前表达量显著升高(P<0.05),在第20天降低至较低水平,25天后表达量再次显著升高,在第30天达到峰值(P<0.05)。研究结果首次揭示了MSTN基因在黄条鰤的胚胎及早期生长发育过程中发挥着重要作用,本研究为开展黄条鰤繁养殖、育种提供了理论参考。

Myostatin(MSTN)is a member of the transforming growth factor-β(TGF-β)superfamily that functions as a negative regulator in the muscle growth and development of animals.To understand the regulatory roles of MSTN in the growth and development of S.aureovittata,the full-length cDNA sequences of MSTN was isolated with the rapid amplification of cDNA ends(RACE)method.The real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expression of MSTN in different tissues,at embryonic development stages,and at larval and juvenile stages in this study.The full length cDNA was1828 bp in length and the open reading frame was 1131 bp(GenBank accession number,MH144053)which encoded 376 amino acids.The putative MSTN of S.aureovittata had the essential characteristic of vertebrate MSTN.qRT-PCR analysis demonstrated that the MSTN differentially expressed in brain,pituitary,liver,muscle,spleen,kidney,gill,heart,stomach,intestine and head-kidney of adult S.aureovittata with the highest in muscle(P<0.05).The MSTN was confirmed to express at every stage of embryogenesis of S.aureovittata.During the embryonic development,the expression of MSTN increased significantly in the embryo encircling 2/3 of yolk sac,and peaked in the hatching larva(P<0.05).At the larval and juvenile stages,the expression of MSTN was significantly up-regulated at 3 days post-hatching(dph),then decreased to a lower level at 20 dph(P<0.05).The expression of MSTN significantly increased at 25 dph and peaked at 30 dph(P<0.05).Taken toget-her,these results provided the first evidence that MSTN plays important regulatory roles at embryo and early developmental stage and also a theoretical basis for reproduction,culture and breeding of S.aureovittata.