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李兵部, 曹哲明, 陶易凡, 徐钢春, 许爱国, 李鸣霄, 张红英, 陈诗扬, 强俊, 徐跑. 大口黑鲈amh基因全长cDNA克隆、表达及多克隆抗体制备[J]. 水生生物学报, 2023, 47(5): 775-785. DOI: 10.7541/2022.2021.0343
引用本文: 李兵部, 曹哲明, 陶易凡, 徐钢春, 许爱国, 李鸣霄, 张红英, 陈诗扬, 强俊, 徐跑. 大口黑鲈amh基因全长cDNA克隆、表达及多克隆抗体制备[J]. 水生生物学报, 2023, 47(5): 775-785. DOI: 10.7541/2022.2021.0343
LI Bing-Bu, CAO Zhe-Ming, TAO Yi-Fan, XU Gang-Chun, XU Ai-Guo, LI Ming-Xiao, ZHANG Hong-Ying, CHEN Shi-Yang, QIANG Jun, XU Pao. MOLECULAR CLONING, EXPRESSION ANALYSIS AND POLYCLONAL ANTIBODY PREPARATION OF ANTI-MÜLLERIAN HORMONE (amh) GENE IN LARGEMOUTH BASS MICROPTERUS SALMOIDES[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(5): 775-785. DOI: 10.7541/2022.2021.0343
Citation: LI Bing-Bu, CAO Zhe-Ming, TAO Yi-Fan, XU Gang-Chun, XU Ai-Guo, LI Ming-Xiao, ZHANG Hong-Ying, CHEN Shi-Yang, QIANG Jun, XU Pao. MOLECULAR CLONING, EXPRESSION ANALYSIS AND POLYCLONAL ANTIBODY PREPARATION OF ANTI-MÜLLERIAN HORMONE (amh) GENE IN LARGEMOUTH BASS MICROPTERUS SALMOIDES[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(5): 775-785. DOI: 10.7541/2022.2021.0343

大口黑鲈amh基因全长cDNA克隆、表达及多克隆抗体制备

MOLECULAR CLONING, EXPRESSION ANALYSIS AND POLYCLONAL ANTIBODY PREPARATION OF ANTI-MÜLLERIAN HORMONE (amh) GENE IN LARGEMOUTH BASS MICROPTERUS SALMOIDES

  • 摘要: 为研究大口黑鲈(Micropterus salmoides)抗缪勒氏管激素(amh)基因的表达及其在性腺发育中的潜在作用, 研究利用RACE技术克隆得到了大口黑鲈amh基因, 并制备Amh多克隆抗体, 通过qRT-PCR、Western Blot分析Amh在大口黑鲈不同组织和不同发育阶段性腺中的表达模式, 最后利用HE染色法和免疫组化观察不同发育阶段性腺的形态组织学变化及其与Amh表达的潜在关系。结果显示: 大口黑鲈amh基因 cDNA序列全长2050 bp, 由24 bp5′非编码区、394 bp3′非编码区和1632 bp的开放阅读框组成, 共编码543个氨基酸。amh基因mRNA在大口黑鲈11个组织中均有表达, 其中雄鱼精巢中表达量最高, 肌肉次之, 雌鱼卵巢中表达量最高, 肌肉次之。amh基因在雌雄鱼不同发育阶段的性腺中表达存在显著差异, 精巢中表达量均显著高于卵巢(P<0.05)。同时, Western Blot结果显示Amh蛋白在精巢中表达丰度较高。amh基因在精巢中的表达量呈先上升后下降的趋势, 且在孵化后65d鱼精巢中其表达量达到最高(P<0.05), 免疫组化结果显示Amh表达于早期精巢的支持细胞; 孵化后135d和215d雄鱼精巢中其表达量明显下降, HE染色显示雄鱼精巢处于精子细胞生成和精子形变期, Amh主要表达于支持细胞中, 精子占据精巢的绝大部分空间, 支持细胞所占面积比例减少。amh基因在孵化后45d、65d和135d雌鱼卵巢中表达量较低, 孵化后215d表达量上升, 并且在卵母细胞的细胞膜边缘及外周的滤泡细胞和颗粒细胞检测到了Amh阳性信号。研究结果表明: 在精巢中, Amh可能参与早期精巢发育; 在卵巢中, 其可能参与颗粒细胞和滤泡细胞发育。综上所述, Amh在大口黑鲈性腺发育中发挥着关键的调控作用。

     

    Abstract: In order to study the characteristics of amh gene in largemouth bass Micropterus salmoides and its potential role during gonadal development, the cDNA full-length sequence of the amh gene was cloned by RACE technology, and the Amh polyclonal antibody was prepared. Quantitative real-time PCR (qRT-PCR) and Western Blot technology was used to analyze the expression pattern of amh in different tissues and different developmental stages. Finally, HE staining and immunohistochemistry were used to observe the morphological and histological changes of gonads at different developmental stages and their potential relationship with Amh expression. The results showed that the cDNA full-length sequence of the amh gene in largemouth bass was 2050 bp in length, including 24 bp at the 5′-UTR, 394 bp at the 3′-UTR, and a 1632 bp open reading frame (ORF) encoding 543 amino acids. The amh mRNA was found expressed in eleven tissues. The expression level in testis was the highest for male fish, then followed by muscle, and the expression level in ovary was the highest for female fish, followed by muscle. There were significant differences in the expression of amh gene in gonads of male and female fish at different developmental stages, and the expression in testis was significantly higher than that in ovary (P<0.05). Western Blot showed that Amh protein was highly expressed in testis. The expression in testis increased first and then decreased, and reached the highest after hatching 65d (P<0.05). Immunohistochemical results showed that Amh was expressed in Sertoli cells of early testis; the expression in male testis decreased significantly at 135d and 215d after hatching. HE staining showed that the testis of male fish was in the stage of spermatogenesis and sperm deformation, Amh was mainly expressed in Sertoli cells. Sperm occupied most of the space of testis, and the proportion of Sertoli cells decreased. The expression was low in female ovaries at 45d, 65d and 135d after hatching, and increased at 215d after hatching, and Amh positive signals were detected in the edge of oocyte membrane and peripheral follicular cells and granulosa cells. The results showed that Amh gene may be involved in the early testicular development in testis; in the ovary, it may participate in the development of granulosa cells and follicular cells. In conclusion, Amh played a key regulatory role in gonadal development of Micropterus salmoides.

     

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