斜带石斑鱼生长激素/催乳素家族基因cDNAs的分子克隆及其进化意义

Molecular Cloning and Evolutionary Implications of Growth Hormone/Prolactin Family Gene cDNAs in Grouper Epinephelus coioides

从斜带石斑鱼垂体提取总RNA ,再取其 5 0ng合成SMARTcDNA。从所构建的垂体SMARTcDNA质粒文库中筛选到生长激素 /催乳素基因家族的 2个成员的全长cDNA片段 :生长激素 (GH )基因全长为 938bp ,编码 2 0 4个氨基酸 ;催乳素基因 (PRL)全长为 14 2 9bp ,编码 2 12个氨基酸。采用计算机软件Mega 2和CLUSTALW1 6 4b对 9种鱼的生长激素 /催乳素基因家族的 3个成员 (GH、PRL和生长催乳素SL)的氨基酸序列进行系统分析 ,构建NJ分支系统树 ,对于序列中的插入 /缺失位点则采用PairaiseDeletion ,10 0 0次自展(Bootstrap)分析计算各节点支持率。根据 3个基因的氨基酸序列构建的系统树表明 ,石斑鱼与金头鲷、金鲈和牙鲆聚成一类 ,虹鳟与大马哈鱼聚成一类 ,鲫鱼与鲶鱼聚成一类 ,鳗鲡成另外一类。根据石斑鱼全长cDNA推断的氨基酸序列比较表明 ,SL相对GH和PRL有较高的保守性。石斑鱼的GH、PRL和SL的氨基酸同源性在2 4 %～ 31% ,但其C -端的氨基酸同源性较高 ,尤其是C -端的 3个Cys是严格保守的。其中SL与GH的同源性 (30 8% )高于与PRL的同源性 (2 5 6 % ) ,GH和PRL的同源性最低 (2 4 1% )。

Total RNA was isolated from pituitary of grouper (Epinephelus coioides),and SMART cDNA was synthesized from 50 ng RNA.Two genes in the growth hormone/prolactin family were cloned from SMART cDNAs plasmid library.The growth hormone (GH) cDNA contains 938?bp and encodes 204 amino acids (aa).The prolactin (PRL) cDNA contains 1?429?bp and encodes 212 aa.The deduced aa sequences of grouper GH,PRL and SL were compared with those of other eight species.Average genetic distances and a dendrogram showing the hierarchical structure of affinity among different nine fishes were implemented in Mega 2 and CLUSTAL W1.64b.Branch and bound strap analysis (1000 replicates) were performed and the degree of support for particular nodes were produced.The three phylogenetic trees drawn from the whole aa sequence of GH,PRL and SL were almost identical,Epinephelus coioides,Perca flavescens,Sparus aurata and Paralichthys olivaceus were clustered into one group,Oncorhynchus keta and Oncorhynchus mykiss were clustered into one group,Carassius auratus and Ictalurus punctatus were clustered into one group,while Anguilla japonica alone was clustered into another group.Alignment of the deduced aa sequence and the phylogenetic tree based on GH/PRL/SL showed the same clustering as the present hierarchy of fish and a higher conservation of SL than GH and PRL among various fish species is suggested in this text.Although there is only 24%-31% identity between the three hormones overall,the C-terminal region shows a higher identity.Grouper SL appears to be more closely related to GH (30.8%) than to PRL (25.6%),which is slightly more than the identity of GH to PRL (24.1%).