摘 要: 采用同源克隆与SMART RACE技术首次分离和克隆了翘嘴鳜性腺型芳香化酶基因CYP19a的cDNA全长, 并通过实时荧光定量PCR (real-time quantitative PCR, qPCR)技术分析了其在翘嘴鳜成体不同组织及性腺不同发育时期的表达特点。序列分析结果显示, CYP19a基因全长1 873 bp (GenBank登录号为KX911988), 其中开放阅读框长1 581 bp, 编码526个氨基酸。通过多序列比对, 发现该基因编码的蛋白质具有P450aromA特定的功能保守序列, 包括I-螺旋区、Ozol肽区、亚铁血红素结合区域以及芳香化酶特异性结合区域; 同时, CYP19a基因序列的同源性分析结果显示, 其在脊椎动物中较为保守。此外, qPCR检测结果显示, CYP19a基因在翘嘴鳜成体组织中的表达存在显著差异(P<0.05), 主要在性腺中表达, 其次在脑和肝脏有少量表达; 而且CYP19a基因在繁殖期性腺中的表达量显著低于非繁殖期性腺中的表达量(P<0.05)。以上研究表明, CYP19a基因在鱼类性腺形成及发育过程中具有重要作用。

Abstract: By using the homology-based cloning combined with rapid-amplification of cDNA ends (RACE), the full length cDNA sequence of gonad-specific aromatase (P450aromA) gene CYP19a from Mandarin fish (Siniperca chuatsi) was obtained. The real-time quantitative PCR (qPCR) was used to detect the relative mRNA expression levels of CYP19a in different tissues and gonads at different development periods. The data showed that the full length of goldfish CYP19a cDNA was 1 873 bp (GenBank accession number KX911988), with an open reading frame of 1 581 bp encoding a protein of 526 amino acids, which contains the transmembrane helix region, I-helix region, Ozol’s peptide region, aromatase-specific conserved region and heme-binding region. Meanwhile, homologous analysis of CYP19a sequences revealed that the gene was more conservative in vertebrates. In addition, the qPCR analysis showed that CYP19a transcripts were highly abundant in ovary and testis (P<0.05), but less abundant in brain and liver. The relative mRNA expression level of CYP19a in pre-reproductive period of gonad was significantly higher than that in reproductive period (P<0.05). Taken together, the results indicate that CYP19a plays an important role in fish gonad formation and developmental processes.