金钱鱼抗缪勒氏管激素基因克隆及其在性腺发育不同时期mRNA表达水平的分析

Gene cloning of anti-Müllerian hormone and analysis of its mRNA level in Scatophagus argus at the different development stages of gonad

为了解抗缪勒氏管激素基因（AMH）在金钱鱼性腺发育中的作用,本研究利用RACE技术克隆了AMH的cDNA序列全长,为2 324 bp（Gen Bank登录号：KP718479）,其开放阅读框为1 631 bp,编码543个氨基酸。同源性分析显示金钱鱼AMH与花鲈相似性最高,为71.16%,与斑马鱼相似性仅为29.83%。系统进化树分析表明,该基因与鲈形目紧密聚为一支,与金钱鱼进化地位一致,说明AMH在进化中有一定保守性。氨基酸结构分析表明其1-28为信号肽序列,69-426为AM H-N区域,444-543为TGF-β结构区。实时荧光定量研究表明,金钱鱼成鱼中AMH基因在精巢中表达量显著高于其他组织,在肝和卵巢中也有表达。性腺不同发育时期分析表明,AMH基因在精巢发育Ⅰ、Ⅱ和Ⅲ期均维持高水平表达,IV期表达水平有所降低,H.E染色结果显示这一时期精巢发育逐渐成熟,推测该基因在精巢发育和精子产生过程中有重要作用。在卵巢中,AMH在Ⅰ、Ⅱ期卵巢发育初期表达量较低,在Ⅲ、Ⅳ期卵母细胞大生长期及卵黄积累期表达量升高,推测其在卵母细胞的发育和功能维持上发挥作用。提示AMH基因在金钱鱼精巢、卵巢发育过程中均发挥重要作用。

To study the role of AMH in gonadal development of Scatophagus argus,we cloned the full-length c DNA sequence of antik-Müllerian hormone（ AMH） from S. argus using the RACE technique,which is 2 324 bp in length（ Gen Bank no. ： KP718479）. The open reading frame（ ORF） of this gene spanned a region of 1 631 bp and encoded 543 amino acids. AMH of S. argus shared 71. 16% similarity in sequence with that of Lateolabrax japonicus,while only 29. 83% similarity with that of Danio rerio. S. alignment analysis revealed that S. argus AMH is closely related to Perciformes AMH,indicating AMH is conserved during evolution. The protein peptide contains a signal peptide（ 1-28 amino acids）,an AMH-N domain（ 69-426 amino acids）,and a TGF-β conserved domain（ 444-543 amino acids）. Results of Real-time PCR showed that the mRNA level of AMH in testis of adult S. argus is significantly higher than that in other organs. AMH expression was also detected in liver and ovary. In addition,the mRNA expression of testis AMH at the development stages of I,II and III was kept at a high level,but was significantly decreased at the development stage of IV. According to the results of H. E staining,spermatogenesis happened in these stages,indicating an important role of AMH in testis development and the sperm production. In the ovary,AMH expression level in Ⅰ,Ⅱ oocyte development stages is low,while in the Ⅲ,Ⅳ stages,when the oocytes are rapidly growing and yolk are gradually accumulating,the AMH level is high. The results demonstrated that AMH is involved in the development and function maintaining of oocyte. Taken together,AMH played a significant role in the development and function of both testis and ovary.