7种鲽形目鱼类生长激素基因cDNA序列的克隆及系统分析

Molecular Cloning and Sequence Analysis of the Growth Hormone cDNAs from Seven Pleuronectiformes Fishes

从7种重要鲽形目经济鱼类——高眼鲽（Cleisthenes herzensteini）、黄盖鲽（Limanda yokohamae）、木叶鲽（Pleuronichthys Cornutus）、宽体舌鳎（Cynoglossus robustus）、石鲽（Platichthys bicoloratus）和条鳎（Zebrias zebra）、夏鲆（Paralichthys dentatus）的脑垂体中提取mRNA，通过RTPCR方法，克隆了含开放阅读框的生长激素（growth hormone，GH）cDNA序列，与本实验室已克隆的大菱鲆和漠斑牙鲆、已报道的鲆鲽鱼类及外群鱼类共22条生长激素基因cDNA进行了序列比对和同源性分析．测序结果显示，高眼鲽、黄盖鲽、木叶鲽、宽体舌鳎、石鲽、条鳎和夏鲆的GHeDNA长度依次为479，564，519，440，564，440，522bp，编码140～170个氨基酸的成熟GH多肽片段．通过序列比对，这7种蛋白序列与其他已知的鲆鲽鱼类GH序列具有较高的同源性，同科的鱼类之间同源性更高．运用PAUP软件对15种鲽形目鱼类与另外7种不同种属鱼类进行了分子系统进化树分析，结果与根据传统的形态学和生化特征分类进化地位基本一致，大菱鲆和塞内加尔鳎各自单独形成一个分支且与鲆科和鲽科鱼类相距较远，其中寒内加尔鳎等种类的分类地位与根据线粒体DNA序列分析的系统发生模式基本吻合．说明生长激素基因可以有效地用于研究鲽形目等硬骨鱼类的亲缘关系及分类地位．

The growth hormone （GH） gene has been cloned from a number of fishes and was used to clarify phylogenetic relationships. The mRNA of Pituitary glands from seven important economic Pleuronectiformes Fishes was extracted, the cDNA sequences of GH gene were isolated from Cleisthenes herzensteini , Limanda yokohamae , Pleuronichth ys Cornutus , Cynoglossus robustus , Platichth ys bicoloratus,Zebrias zebra and Paralichthys dentatus with RT-PCR method by using designed specific primers based on the reported GH cDNA sequences of Pleuronectiformes. Alignment and homologous analysis of All 7 GH cDNA sequences was performed with other 8 Pleuronectiformes fishes and 7 outgroup species. The result of sequencing showed the lengths of the above cDNAs as follows, 479, 564, 519, 440, 564, 440 and 522 bp, and the GH cDNA sequences encoding mature polypeptide of about 140 to 170 amino acid. The molecular weight and isoelectric point of GH protein from Platichthys bicoloratus ,Limanda yokohamae,Pleuronichthys Cornutus and Paralichthys dentatus were calculated with software DNASTAR 7. 0. After alignment of 7 GH with other Pleuronectiformes fishes, the 7 GH amino acid sequences all showed high homology to other known Pleuronectiformes fishes, especially to the sequences within the same family. Then the phylogenetic analysis on the GH for 15 species of Pleuronectiformes and 7 outgroup species was performed by PAUP software with the maximum parsimony method. The result was consistent with morphology of Pleuronectiformes on the whole. In the topology founded, Scophthalmus maximus and Solea senegalensis each formed an independent branch and showed far relationship with species of Bothidae and Pleuronectidae. The phlogenic position of Solea senegalensis is in accord with the result of analysis made by mitochondrial genome. It seems reasonable to speculate that GHs can be effectively used in analyzing genetic relationship and taxonomic status of Pleuronectiformes and other species of teleostean.