双须骨舌鱼抗缪勒氏管激素基因amh的克隆、组织表达分析和原核表达

Molecular cloning, expression pattern and prokaryotic expression of anti-Müllerian hormone (amh) gene in Osteoglossum bicirrostum

摘要:

为探究双须骨舌鱼抗缪勒氏管激素基因amh的作用和表达模式，本研究通过RACE技术克隆了双须骨舌鱼amh基因全长cDNA序列，发现存在amh1、amh2两个亚型（GenBank登录号KU378662、KU378663）。序列分析结果显示，双须骨舌鱼amh1基因的cDNA全长2277 bp，编码623个氨基酸，amh2基因cDNA全长2181 bp，编码355个氨基酸。同源性分析显示，amh基因保守性较低，双须骨舌鱼与同科的美丽硬仆骨舌鱼相似度最高，为85.64%，与不同科鱼类的相似度均低于42%。系统进化分析显示，该基因与骨舌鱼目聚为一支，与鲱形目、鲤形目等较低等的硬骨鱼类亲缘关系较近，与双须骨舌鱼进化地位相符。用荧光定量PCR检测的结果显示，amh基因在双须骨舌鱼不同组织中均有表达，其中amh1亚型在精巢中表达量最高，且明显高于卵巢和其他组织；amh2亚型也在精巢中表达量最高，且明显高于其他组织，但远远低于amh1在精巢中的表达。构建了原核表达载体pColdⅠ-amh1和pColdⅠ-amh2并在大肠杆菌中成功诱导出大小分别为68和48 ku的融合蛋白，为进一步研究amh在双须骨舌鱼体内的生物功能奠定了基础。

关键词:双须骨舌鱼;抗缪勒氏管激素;克隆;表达分析;原核表达

Abstract:

In order to explore the function and expression pattern of anti-Müllerian hormone (amh) gene in Osteoglossum bicirrostum, this study cloned the whole cDNA of amh gene in O. bicirrostum using RACE method, and found that O. bicirrostum had two different hypotypes——amh1 and amh 2(Genbank no.:KU378662, KU378663) of this gene. The result of sequence analysis showed that Amh1 had the full-length of 2277 bp, and encoded 623 amino acids; Amh2 had the full-length of 2181 bp, and encoded 355 amino acids. The homology analysis showed that the Amh in O. bicirrostum shared little conservation, and the similarity compared with its coordinal Scleropages formosus was the highest (85.64%), but that with fishes in other families was lower than 42%. Phylogenetic analysis showed that Amh came to be a cluster with Osteoglossiformes Amh and was closely related with lower teleost species such as Clupeiformes and Cypriniformes, which agreed with the evolutionary position of O. bicirrostum. The real-time fluorescent quantitative PCR results showed that amh was expressed generally in many tissues. The expression of amh1 was the highest in the testis, and much higher than that in ovary and other tissues. The expression of amh2 was also the highest in the testis, and much higher than that in other tissues, but much lower than the expression of amh1 in the testis. We constructed prokaryotic expression vectors pColdⅠ-amh1 and pColdⅠ-amh2, and successfully induced proteins of 68 and 48 ku in Escherichia coli, which laid the foundation for further study of amh in O. bicirrostum.

Key words:Osteoglossum bicirrostum;anti-Müllerian hormone;clone;expression analysis;prokaryotic expression