淇河鲫cyp19a1b基因的克隆表达及芳香化酶抑制剂对其表达的影响
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河南师范大学,河南师范大学,河南师范大学,河南师范大学,河南师范大学

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国家自然科学基金(31702316,31602149);河南省科技攻关计划项目(172102210351,152102110082);河南省高等学校重点科研项目(15A240002,18A240002);河南省自然科学基金(162300410164);河南师范大学博士科研启动基金(qd16158)


Molecular cloning and characterization of cyp19a1b gene and the effect of Letrozole on its expression in Carassius auratus
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Henan Normal University,Henan Normal University,Henan Normal University,Henan Normal University,Henan Normal University

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    摘要:

    为探索脑型芳香化酶基因(cyp19a1b)在雌核发育三倍体鱼淇河鲫性别决定与分化过程中的作用,利用RACE方法克隆淇河鲫cyp19a1b基因cDNA全长序列,采用Real-time PCR分析其在不同组织、胚胎及胚后不同发育时期的表达情况,同时检测芳香化酶抑制剂Letrozole诱导性逆转及腹腔注射人绒毛膜促性腺激素(hCG)后cyp19a1b在脑中的表达情况。结果显示,淇河鲫cyp19a1b cDNA全长2 984 bp (GenBank ID: MF926270),包含132 bp 5'非编码区,1 319 bp 3'非编码区,1 533 bp开放读码框,编码510个氨基酸残基;氨基酸序列比对及系统进化分析结果显示,淇河鲫Cyp19a1b与其他鲤科鱼类同源性较高,与哺乳类、爬行类等脊椎动物同源性较低,这与其分类地位一致;组织分布检测结果显示,cyp19a1b基因在淇河鲫脑中表达量最高,在卵巢等其他组织中表达量较低;Real-time PCR结果表明,在胚胎发育过程中cyp19a1b在外源精子刺激后从囊胚期开始上调,神经胚期达到最高,随后降低;出膜后随着发育的进行,该基因在脑中表达量逐步上调,在躯体中一直维持在较低水平;除此之外,伴随着Letrozole诱导的性逆转,cyp19a1b在脑中表达量降低;腹腔注射hCG可以促进cyp19a1b在脑中表达。研究表明,淇河鲫cyp19a1b基因可能通过参与神经系统的形成及神经内分泌活动,在性别决定与分化过程中起到一定的作用。

    Abstract:

    In the present study, brain aromatase gene (cyp19a1b) was cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA 5' and 3' ends (RACE) in Qihe crucian carp (Carassius auratus). We also examined the expression level of cyp19a1b in different development stages and different tissues by real-time PCR, and at the same time, expression profile of cyp19a1b after hCG injection and sex reversal induced by Letrozole treatment was also examined. The results showed that the full-length cDNA of cyp19a1b was 2 894 bp, including a 132 bp 5'-untranslated region (UTR), a 1 319 bp 3'UTR, and a 1 533 bp open reading frame (ORF) encoding a deduced protein with 510 amino acids. Sequence analysis revealed that Cyp19a1b from C. auratus showed the highest amino acid similarity with Cyprinidae fish, while has low similarity with other vertebrates include mammalian and reptiles. Tissue distribution by real-time PCR revealed that cyp19a1b was abundantly expressed in the brain, while barely detectable in other tissues. The expression of cyp19a1b was gradually upregulated from blastula stage, and peaked at neurula stage, then declined during the embryonic development stage. Ontogenic analysis demonstrated that cyp19a1b was expressed abundantly from 30 days after hatching (dah) in the brain, but kept a low level in the trunks. However, cyp19a1b decreased in the brain of sex-reversed males by Letrozole treatment. Intraperitoneal injection of human chorionic gonadotropin experiments showed that expression of cyp19a1b was significantly elevated at 8 h and persisted until 24 h after injection in the brain. These results suggested that cyp19a1b might be involved in the course of nervous system formation, gonad development and sex determination in C. auratus.

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李永婧,吴利敏,王磊,马晓,李学军.淇河鲫cyp19a1b基因的克隆表达及芳香化酶抑制剂对其表达的影响[J].水产学报,2018,42(8):1169~1180

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  • 收稿日期:2017-09-11
  • 最后修改日期:2018-01-09
  • 录用日期:2018-03-04
  • 在线发布日期: 2018-08-01
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