20β-hydroxysteroid dehydrogenase and CYP19A1 are differentially expressed during maturation in Atlantic cod (Gadus morhua)
20β-hydroxysteroid dehydrogenase and CYP19A1 are differentially expressed during maturation in Atlantic cod (Gadus morhua)
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DOI：
10.1677/jme-07-0070
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发表时间：
2007-09-01
影响因子：
3.5
通讯作者：
Norberg, B.
中科院分区：
文献类型：
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作者：
Mittelholzer, C.;Andersson, E.;Norberg, B.
为了更好地量化调控最终卵母细胞成熟和产卵的分子机制，完整的编码序列与类固醇生成酶，细胞色素P450芳香化酶和20 β-羟基类固醇脱氢酶的部分或全部非翻译区，从大西洋鳕鱼（Gadus morhua）卵巢克隆。的核苷酸和氨基酸序列显示出较高的同源性与其他鱼类的相应序列，和保守功能的重要功能被确定在这两个预测的蛋白质。还确定了相应基因组位点的序列，从而可以设计mRNA特异性定量PCR测定。选择真核细胞延伸因子1 α作为实时RT-PCR测定的参考基因，并测定mRNA和基因组序列。此外，18 S rRNA的实时定量PCR检测适用于鳕鱼。对7月至1月未成熟和成熟雌鳕的分析表明，酶基因表现出与生理调节相关的预期数量变化。然而，真核细胞延伸因子1a的mRNA，甚至在较小程度上18 S rRNA，在这些样品中也显示出可变的表达。在鳕鱼卵巢这样一个动态器官中找到准确的实时PCR标准品并不是一件容易的事情，并讨论了几种可能的解决方案。
In order to better quantify the molecular mechanisms regulating final oocyte maturation and spawning, complete coding sequences with partially or fully untranslated regions for the steroidogenic enzymes, cytochrome P450 aromatase and 20 beta-hydroxysteroid dehydrogenase, were cloned from ovaries of Atlantic cod (Gadus morhua). The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species, and conserved features important for functionality were identified in both predicted proteins. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-specific quantitative PCR assays. As a reference gene for the real-time RT-PCR assays, eukaryotic elongation factor 1 alpha was chosen, and the mRNA as well as the genomic sequence was determined. In addition, a real-time quantitative PCR assay for the 18S rRNA was adapted to be used in cod. Analysis of immature and maturing female cod from July to January respectively showed that the enzyme genes showed the expected quantitative changes associated with physiological regulation. However, mRNA for eukaryotic elongation factor 1 a, and to a lesser extent even 18S rRNA, showed variable expression in these samples as well. To find accurate standards for real-time PCR in such a dynamic organ as the cod ovary is not an easy task, and several possible solutions are discussed.