• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

兰州鲇性腺型芳香化酶cyp19a1a基因克隆及表达定位分析

Cloning and Localization of Gonadal Aromatase Gene in Silurus lanzhouensis

  • 摘要:
      目的  性腺型芳香化酶基因cyp19a1a在硬骨鱼类性腺发育和性别决定过程中具有重要作用,克隆分析该基因在生长和体型性状具有性别二态性兰州鲇(Silurus lanzhouensis)不同组织的表达与定位,并对已获得YY超雄个体的雌雄同体亲本进行不同组织表达验证分析,为加快培育全雄兰州鲇良种新品种提供理论依据和技术支撑。
      方法  采用同源克隆和cDNA末端快速扩增(RACE)法获得兰州鲇cyp19a1a全长cDNA序列,采用实时荧光定量PCR(qRT-PCR)分别检测该基因在3月龄、1龄和3龄兰州鲇正常雌雄鱼和3龄雌雄同体鱼的不同组织表达,并采用免疫组织化学法(IHC)分别检测该基因在3月龄、1龄和3龄正常雌性鱼性腺组织的表达与定位。
      结果  cyp19a1a cDNA序列全长为2168 bp,其中5′ 非编码区(Untranslated region,UTR)53 bp,开放阅读框(OFR)1707 bp,3′ UTR 408 bp,编码568个氨基酸残基,具有芳香化酶氨基酸序列的保守功能区。兰州鲇cyp19a1a mRNA主要在性腺表达,3龄雌雄同体表达特征与3龄正常繁育个体相同,且卵巢表达量显著高于精巢(P < 0.05);正常繁育养殖个体表达量随性腺发育而显著增加。IHC结果显示,不同时期卵巢组织中,卵原细胞仅在3月龄和1龄分布且没有发现阳性信号,卵母细胞在不同时期均有分布且信号随着卵母细胞发育而逐渐增强,其中3月龄至1龄主要分布于胞质,3龄时主要分布于胞质、滤泡膜以及放射膜和鞘膜细胞;不同时期精巢组织中仅在间质细胞有微弱表达。
      结论  cyp19a1a基因在兰州鲇卵巢发育和卵细胞生长发育过程中都具有重要作用,对精巢发育和维持以及精子发生具有一定的促进作用,在雌雄同体兰州鲇性腺中的作用和兰州鲇正常繁育个体一致。本研究为鱼类性别决定与分化以及性腺发育调控机制提供了重要的理论依据。

     

    Abstract:
      Objective  Expression and location of cyp19a1a important in the gonadal development and sex determination of bony fishes were studied to pave the way for breeding all-male Silurus lanzhouensis.
      Method  The gene in the sexual organs of S. lanzhouensis were cloned, and expression of hermaphroditic parents of YY super-male individuals verified. The full-length cDNA sequence of cyp19a1a was obtained by homologous cloning and rapid amplification of cDNA ends (RACE). Expressions of the gene in 3-month-old, 1-year-old, and 3-year-old normal female and male as well as 3-year-old hermaphroditic fish were determined by real-time fluorescence quantitative PCR (qRT-PCR). Expression and localization of the gene in tissues of 3-month-old, 1-year-old, and 3-year-old normal female fish were determined using immunohistochemistry (IHC).
      Result   The full length of cyp19a1a cDNA was 2168 bp, which included 5' untranslated region (UTR) of 53 bp, open reading frame (OFR) of 1707 bp, and 3' UTR of 408 bp, encoding 568 amino acid residues with a conserved functional region of aromatase. Cyp19a1a mRNA was mainly expressed in the gonads. The expressions in 3-year-old hermaphroditic and normal individuals were not significantly different (P>0.05), but significantly higher in ovary than in testis, and significantly intensified along with the development of gonad in the bred normal fish. The IHC showed the oogonia distributed only in the ovaries of 3-month and 1-year-old fish at different stages with no positive signals. Whereas the oocytes were found during all development periods with a gradually increasing signal. They existed mainly in the cytoplasm from 3-month to 1-year-old, but in the 3-year-old fish in the follicular membrane, radiation membrane, and sheath cells, in addition to cytoplasm. On the other hand, cyp19a1a was only weakly expressed in the interstitial cells of the testis at different developmental stages of a fish.
      Conclusion   There was a significant role cyp19a1a played in the ovarian development, oocyte growth, and oogenesis. It involved in the testicular development and maintenance and spermatogenesis of normal or hermaphroditic S. lanzhouensis identically.

     

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