| 摘要: |
| 为了探讨Cyp19a1b基因表达与温度及鱼油添加量的相互关系, 奠定金钱鱼人工繁育理论基础,作者采用RT-PCR 和RACE 法, 克隆了金钱鱼(Scatophagus argue Linnaeus)脑型芳香化酶基因(Cyp19a1b); 采用实时荧光定量PCR 法, 检测了不同温度(23、26 和29℃)和不同水平鱼油(0、2%和6%)处理后二龄雌性金钱鱼脑垂体中Cyp19a1b mRNA 表达。结果表明: Cyp19a1b cDNA 全长2409bp, 5′端非编码区164 bp, 3′端(不包括poly(A))712 bp, 开放阅读框(ORF)1506 bp, 编码501 个氨基酸, 推测其蛋白质分子量为56.697 kDa。序列分析及分子系统进化树结果表明: 金钱鱼Cyp19a1b 氨基酸序列与黄锡鲷和舌齿鲈Cyp19a1b 同源性较高, 分别为86.2%、86.5%, 与鲤鱼(Cyprinus carpio)、稀有■鲫(Gobiocypris rarus)、斑马鱼(Danio rerio)、赤点石斑鱼(Epinephelus akaara)、大黄鱼(Larimichthys crocea)、南方大口鲇(Silurus meridionalis)和鲻鱼(Mugil cephalus)Cyp19a1b同源性为64.1%～84.4%, 但与上述7 种鱼性腺芳香化酶(Cyp19a1a)同源性低于63.5%, 同源性分析结果与根据传统形态学和生化特征分类的结果相一致。荧光定量PCR 结果显示, 随处理时间的延长, 26℃和29℃组金钱鱼脑垂体中Cyp19a1b mRNA 的表达量都逐渐降低, 但29℃组显著低于26℃组(P<0.05), 而23℃组在6 周时则显著高于26℃和29℃组(P<0.05), 约为26℃组2 倍的表达量; 鱼油处理3 个投喂组脑垂体中Cyp19a1b mRNA 的表达量均逐渐降低, 但鱼油对金钱鱼脑垂体中Cyp19a1b mRNA 的表达量无显著影响(P>0.05)。以上结果表明: 随着卵巢发育或鱼油含量的提高, 可降低雌性金钱鱼脑垂体中Cyp19a1b mRNA的表达, 这种降低可能是E2 反馈调节所致; 水温高于26℃将抑制Cyp19a1b mRNA 的表达。 |
| 关键词: 金钱鱼(Scatophagus argue Linnaeus) 温度 鱼油 芳香化酶 mRNA 表达 |
| DOI：10.11759/hykx20130502001 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目(31272640) |
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| Molecular cloning and expression analysis of Cyp19a1b cDNA in spotted scat, Scatophagus argue |
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| Abstract: |
| In this study, in order to investigate the relationship between Cyp19a1b gene expression and the temperature or fish oil amount, laying the theoretical foundation for the artificial breeding of Scatophagus argue, a cDNA encoding Cyp19a1b was derived from S. argue, using RT-PCR and RACE methods. Meanwhile, we investigated the effect of different temperature (23℃, 26℃, and 29℃) and concentration of dietary fish oil (0, 2%, and 6%) on pituitary Cyp19a1b mRNA expression in 2-year-old female S. argue. Results showed that the cDNA was 2409 bp with 164 bp 5′UTR, 712bp 3′UTR (excluding poly (A)) and 1506 bp ORF, which encoded 501 amino acids and had a predicted mol wt of 56.697 kDa. Sequence and phylogenetic analysis indicated that the S. argue Cyp19a1b shared 86.2% and 86.5% sequence identity with Rhabdosargus sarba and Dicentrarchus labrax respectively, and between 64.1%～84.4% with that of Cyprinus carpio, Gobiocypris rarus, Danio rerio, Epinephelus akaara, Larimichthys crocea, Silurus meridionalis, and Mugil cephalus. However, it shared lower sequence identity (below 63.5%) with Cyp19a1a of the fish above. These results were consistent with those analyzed by traditional morphology and biochemistry in their evolution status. The fluorescent real time quantity PCR analysis revealed that pituitary Cyp19a1b mRNA expressions were found gradually decreasing with treatment time both in 26℃ and 29℃ group, but that in 29℃ group was significantly lower than that in 26℃ group (P<0.05). However, higher pituitary Cyp19a1b mRNA expression was found in 23℃ group at 6 weeks compared to that in 26℃ and 29℃ group, and was about 2 times higher than that in 26℃ group. In addition, pituitary Cyp19a1b mRNA expressions were found gradually decreasing with feeding time in 3 groups of different concentration of dietary fish oil, but no significant difference was found among the 3 groups (P>0.05). Results showed that pituitary Cyp19a1b mRNA expression would gradually decease with ovarian development or increasing of fish oil concentration in female S. argue, which might be caused by estradiol feedback regulation, and temperature more than 26℃ would restrain pituitary Cyp19a1b mRNA expression. |
| Key words: Scatophagus argue temperature fish oil Cyp19a1 mRNA expression |
