食蚊鱼CYP19a基因的克隆与序列分析

Molecular cloning and analysis of CYP19a gene in mosquitofish(Gambusia affinis)

硬骨鱼类CYP19基因与生物的性别分化和激素调节相关,因此可开发用来探究环境激素污染与基因表达的关系。本研究首次克隆和分析了食蚊鱼CYP19a cDNA的全系列,为将CYP19基因作为监测环境激素生物标志物的研究提供了全面的实验数据。根据CYP19a基因c DNA保守区域设计引物,扩增保守区域并测序。采用RACE法扩增食蚊鱼CYP19a基因c DNA序列全长,对其蛋白序列进行同源性分析,并将序列应用于CYP19a mRNA转录水平的RT-PCR法检测中。成功克隆食蚊鱼CYP19a基因全长,获得CYP19a基因总长为2020 bp,ORF为238～1791 bp,共编码518个氨基酸,对其编码的蛋白质进行有关信号肽、跨膜螺旋、亲水性/疏水性、一级结构、二级结构和三级结构分析,与其他硬骨鱼类底鰆、青鰆、平鲷、鲫、鲤和斑马鱼的性腺CYP19a基因作同源性比较,其基因相似度分别为93%、84%、84%、71%、71%和66%。用MEGA6.0软件对19个物种的CYP19a基因进行聚类分析,食蚊鱼CYP19a基因与底鰆、青鰆同源性最高,说明芳香化酶在进化上相对保守。确定从食蚊鱼性腺所克隆的CYP19a基因是芳香化酶基因,证明食蚊鱼的芳香化酶是由CYP19a和CYP19b两种基因编码的。食蚊鱼卵巢芳香化酶具有3个高度保守的片段,并具有催化活性。

Biological sex differentiation and hormonal regulation are related with the expression of CYP19 gene in teleost, and so it can be used to explore the relationship between environmental hormone pollution and gene expression. The Gambusia affinis CYP19 a cDNA of full sequence was cloned and analyzed for the first time. This would provide comprehensive experimental data for the study of the CYP19 gene as a biomarker for monitoring environmental hormones. Primers were designed according to the conserved region of CYP19 a cDNA, and the conserved region was amplified and sequenced. RACE method was used to amplify the G. affinis CYP19 a cDNA of full sequence and its protein sequence homology analysis, and the RT-PCR method was used to detect the transcription level of CYP19 a mRNA in the sequence. The full length cDNA sequence of G. affinis CYP19 a type was cloned. This sequence contains 2020 bp nucleotides and codes 518 amino acids with an open reading frame（ORF） from 238 bp to 1791 bp. We made an analysis of the signal peptide, transmembrane helices, hydrophilic/hydrophobic, primary structure, secondary structure and tertiary structure. When making the homology comparison with CYP19 a gene in gonads of other teleost, the gene fragment similarity of mosquitofish were 93%, 84%, 84%,71%, 71% and 66% with Fundulus heteroclitus, Oryzias laticeps, Rhabdosargus sarba, Carassius auratus,Cyprinus carpio and Danio rerio respectively. This showed that the cloned gene was G. affinis CYP19 a gene.Phylogenetic analysis of the CYP19 gene by using MEGA4.0 indicates that CYP19 a gene is highly conserved when clustered with other 19 species ovary-derived P450 arom gene. We identified the CYP19 a cDNA of full sequence is gonadal aromatase gene, and the proof of G. affinis aromatase is by two genes of CYP19 a and CYP19 b encoding. G. affinis CYP19 a has 3 highly conserved fragments, and has catalytic activity.