金钱鱼cyp17a1基因克隆、组织分布及在卵巢发育中的表达

Cloning,Tissue Distribution and Expression of cyp17a1 at Different Ovarian Development Stages in Spotted Scat(Scatophagus argus)

【目的】克隆金钱鱼cyp17a1基因,分析其在组织中的分布、卵巢不同发育时期中的表达变化,为金钱鱼繁殖生物学提供理论依据。【方法】通过金钱鱼转录组文库筛选和RT-PCR进行cyp17a1 c DNA序列扩增;用DNAstar软件比较Cyp17a1同源性,MEGA5.0构建系统进化树,RT-PCR检测cyp17a1组织表达,实时定量PCR检测不同卵巢发育时期cyp17a1的表达量。【结果】金钱鱼cyp17a1开放阅读框编码515个氨基酸残基;Cyp17a1氨基酸序列与同属鲈形目鱼类Cyp17a1同源性较高(87.8%～92.7%),与大黄鱼最高(92.7%),与其他目鱼类同源性次之(72.7%～85.0%),与硬骨鱼类Cyp17a2或Cyp17a1-like同源性最低(48.2%～51.7%);所有硬骨鱼类Cyp17a1聚为一支,在Cyp17a1分支中,金钱鱼与鲈形目鱼类聚为一支;cyp17a1在性腺中表达量较高,在精巢中表达最强;在肝、脑和头肾表达较弱;在脾脏、肠、心脏、肌肉和鳃中未检测到表达;卵巢中cyp17a1的表达量随卵巢发育逐渐增加,Ⅳ期卵巢中cyp17a1表达量最高。【结论】cyp17a1在性腺中表达量较高,在金钱鱼卵巢发育过程中有重要作用。

【Objective】To provide a theoretical basis for the reproductive biology of spotted scat （Scatophagus argus）, the cyp17a1 gene was cloned and analyzed. The tissue distribution and expression of the hypothalamus at different ovarian development stages were also evaluated. 【Methods】The cyp17a1 cDNA was amplified by the combination of transcriptome and RT-PCR. The alignments of the amino acid sequences of Cyp17a1 were performed using DNAstar. A phylogenetic tree was constructed by using MEGA 5.0. Tissue distribution of Cyp17a1 expression was analyzed by RT-PCR. The expression of cyp17a1 at ovarian developmental stages was detected by real-time quantitative PCR.【Results】 The cyp17a1 open reading frame encodes a predicted protein of 515 amino acid （aa） residues. The Cyp17a1 is highly homologous with that of other Cyp17a1 （72.7%–92.7%）, and has low homology with that of Cyp17a2 or Cyp17a1-like （48.2%–51.7%） in teleost. The Cyp17a1 and other teleost Cyp17a1 were clustered into one clade. The spotted scat clustered firmly with other Perciformes fish. RT-PCR showed that the expression of the cyp17a1 was higher in the gonads, with the strongest expression in the testis, but weak in the liver, brain and no expression in head kidney, spleen, intestines, heart, muscle and gills. The cyp17a1 expression increased gradually with ovarian development, and the cyp17a1 transcript level in phase Ⅲ was significantly higher than that in phase II, and the cyp17a1 in phase IV was significantly higher than that in phase Ⅲ. 【Conclusion】 The cyp17a1 has a high expression level in gonads. It plays an important role during the ovarian development in spotted scat.