卵形鲳鲹抗缪勒氏管激素amh基因的克隆、表征及原核表达  

Cloning,Characterization and Prokaryotic Expression of amh Gene in Golden Pompano(Trachinotus ovatus)

作　　者：张智浩 王露皓 赵文栋 刘金相[3] 茅云翔 Zhang Zhihao;Wang Luhao;Zhao Wendong;Liu Jinxiang;Mao Yunxiang(College of Ecology and Environment,Hainan Tropical Ocean University,Sanya 572022,China;Yazhou Bay Innovation Institute,Hainan Tropical Ocean University,Sanya 572024,China;Sanya Oceanographic Institution,Ocean University of China,Sanya 572024,China)

机构地区：[1]海南热带海洋学院生态环境学院,海南三亚572022 [2]海南热带海洋学院崖州湾创新研究院,海南三亚572024 [3]中国海洋大学三亚海洋研究院,海南三亚572024

出　　处：《中国海洋大学学报(自然科学版)》2025年第3期155-168,共14页Periodical of Ocean University of China

基　　金：国家重点研究发展计划项目(2022YFD2400500);三亚崖州湾科技城科技专项(SKJC-2023-01-004)资助。

摘　　要：为探究卵形鲳鲹(Trachinotus ovatus)amh基因的序列特征、组织表达模式和功能作用,本研究从卵形鲳鲹基因组和转录组中获得amh基因的序列并进行了PCR克隆和验证。研究结果显示,卵形鲳鲹amh基因存在三种长度的可变剪切,分别被命名为amh-L、amh-S和amh-C。其中:amh-L和amh-S均具有完整的开放阅读框,编码含有AMH-N结构域和TGF-β结构域的蛋白质;而amh-C由于内含子的保留,导致其阅读框发生了移码并提前终止,无法编码完整的蛋白质。通过系统发育分析发现,卵形鲳鲹的抗缪勒氏管激素(Anti-Müllerian hormone,AMH)与黄条鰤的AMH相似性最高,它们与其他硬骨鱼的AMH聚为一支,与哺乳类等四足动物的AMH明显分开。在组织表达模式方面,本研究发现:在成体卵形鲳鲹体细胞组织中,amh在脑中表达较高,而在其他组织中表达较低,且雌、雄间无明显差异,在性腺中具有明显的性别二态性:精巢中的表达amh显著高于卵巢,随着性腺的发育和成熟,amh基因的表达量逐渐降低;原位杂交结果显示,amh主要表达在早期精巢的支持细胞中,在卵巢中主要分布在早期卵母细胞的细胞质中;利用原核表达系统获得的AMH-L和AMH-S重组蛋白均以包涵体的形式存在于菌体中,纯化后经蛋白免疫印迹(Western blot)检测验证为正确蛋白。本研究为进一步探讨卵形鲳鲹性别分化和性腺发育的分子机制提供基础资料。For clarifying its function,amh gene was identified and cloned from golden pompano(Trachinotus ovatus)genome.Its tissue distribution and expression pattern in gonad at different developmental stages was characterized.We found three spliceosomes of amh in both male and female gonad,named as amh-L,amh-S and amh-C.amh-L was the most abundant type,whereas amh-S and amh-C were relatively rare.amh-L and amh-S contained open reading frame encoding an entire AMH-N domain and a TGF-βdomain which is the typical structure of amh gene.The amh-C was intron retention splicing that retained intron 4 and intron 5,resulting in frame shift mutation and premature termination.Phylogenetic analysis was carried out with AMH amino acid sequences of various species.T.ovatus AMH showed highest similarity with yellow tail and they formed a clade with other teleost species.AMH of tetrapod species grouped into another distinct clade.In somatic tissues amh showed highest expression in brain,but relatively low expression in other tissues.No sex difference was observed.In gonad,however,the expression level of amh showed marked sex dimorphism with testes expressed much higher than ovary.At different development stages amh expression decreased gradually from 6-month to 24-month of age.In situ hybridization analysis found that amh was mainly expressed in the sertoli cells in the testis and the cytoplasm of the oocytes in the ovaries.Two recombinant proteins AMH-L and AMH-S were obtained using prokaryotic expression system with the corresponding molecular weights of the 59 kDa and 55 kDa,respectively.The purified proteins were verified by western blot assay.The results of the present study and the successful expressing of AMH protein provided basic data and material for the further functional study of amh during the gonad differentiation and development in T.ovatus.

关 键 词：卵形鲳鲹 amh基因 剪切体 性别分化 原核表达 

分 类 号：Q492[生物学—生理学]