Huang Wei, Yang Pan, Lv Zhenming, Wu Changwen, Gui Jianfang, Lou Bao
National Engineering Research Center of Marine Facilities Aquaculture, College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan, Zhejiang 316022, China; Key Laboratory of Marine Culture and Enhancement of Zhejiang Province, Zhoushan, Zhejiang 316022, China.
National Engineering Research Center of Marine Facilities Aquaculture, College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan, Zhejiang 316022, China.
Gene. 2017 Sep 5;627:271-277. doi: 10.1016/j.gene.2017.06.042. Epub 2017 Jun 24.
Gonadal-specific aromatase encoded by cyp19a1a is the important enzyme controlling estrogen biosynthesis in teleosts. In the present study, the cDNA sequence of cyp19a1a was cloned and characterized from miiuy croaker Miichthys miiuy. The cDNA encoded a protein of 519 amino acids with five structural regions. Higher identities of amino acid sequences and conserved structural regions were found between Mmcyp19a1a and other cyp19a1a genes. In addition, Mmcyp19a1a was clustered together with other seawater fishes. Immunohistochemical analysis revealed that Mmcyp19a1a was localized exclusively in the cytoplasmic of thecal and granulosa cells surrounding the oocytes. Both the protein and mRNA levels of Mmcyp19a1a were increased significantly at the stage III follicles (mid-vitellogenic) and then decreased along with vitellogenesis. Interestingly, strong immunoreactive signals were also detected in the supporting cells of connective tissues during ovarian development. A 1777bp promoter fragment of Mmcyp19a1a was also isolated, and functional analysis using an EGFP reporter fusion in zebrafish larvae presented positive signals in the above of yolk sac, where is the region of pronephros and germ plasm occur. The Mmcyp19a1a:EGFP expression pattern was generally consistent with the endogenous cyp19a1a genesis. These results indicate that the Mmcyp19a1a gene plays an important role during vitellogenesis and oocyte maturation. The constructor of Mmcyp19a1a:EGFP may provide a useful tool for genetic analysis of gonad development in teleost.
由cyp19a1a编码的性腺特异性芳香化酶是硬骨鱼中控制雌激素生物合成的重要酶。在本研究中，从鮸鱼Miichthys miiuy克隆并鉴定了cyp19a1a的cDNA序列。该cDNA编码一个含有519个氨基酸的蛋白质，具有五个结构区域。在Mmcyp19a1a与其他cyp19a1a基因之间发现了较高的氨基酸序列同一性和保守结构区域。此外，Mmcyp19a1a与其他海水鱼类聚集在一起。免疫组织化学分析显示，Mmcyp19a1a仅定位在卵母细胞周围的膜细胞和颗粒细胞的细胞质中。Mmcyp19a1a的蛋白质和mRNA水平在III期卵泡（卵黄发生中期）显著增加，然后随着卵黄发生而降低。有趣的是，在卵巢发育过程中，结缔组织的支持细胞中也检测到强免疫反应信号。还分离出了Mmcyp19a1a的一个1777bp启动子片段，在斑马鱼幼虫中使用EGFP报告基因融合进行的功能分析在卵黄囊上方呈现阳性信号，那里是前肾和生殖质所在的区域。Mmcyp19a1a:EGFP的表达模式与内源性cyp19a1a的发生基本一致。这些结果表明，Mmcyp19a1a基因在卵黄发生和卵母细胞成熟过程中起重要作用。Mmcyp19a1a:EGFP构建体可能为硬骨鱼性腺发育的遗传分析提供一个有用的工具。
