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

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

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 bp5′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;Realtime 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。

In the present study, brain aromatase gene（cyp19 a1 b） was cloned by using reverse transcription-polymerase chain reaction（RT-PCR） and rapid amplification of c DNA 5′ and 3′ ends（RACE） in Qihe crucian carp（Carassius auratus）. We also examined the expression level of cyp19 a1 b in different development stages and different tissues by real-time PCR, and at the same time, expression profile of cyp19 a1 b after h CG injection and sex reversal induced by Letrozole treatment was also examined. The results showed that the full-length c DNA of cyp19 a1 b 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 Cyp19 a1 b 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 cyp19 a1 b was abundantly expressed in the brain, while barely detectable in other tissues. The expression of cyp19 a1 b was gradually upregulated from blastula stage, and peaked at neurula stage, then declined during the embryonic development stage. Ontogenic analysis demonstrated that cyp19 a1 b was expressed abundantly from 30 days after hatching（dah） in the brain, but kept a low level in the trunks. However, cyp19 a1 b decreased in the brain of sex-reversed males by Letrozole treatment. Intraperitoneal injection of human chorionic gonadotropin experiments showed that expression of cyp19 a1 b was significantly elevated at 8 h and persisted until 24 h after injection in the brain. These results suggested that cyp19 a1 b might be involved in the course of nervous system formation, gonad development and sex determination in C. auratus.