石斑鱼垂体腺苷酸环化酶激活多肽和生长激素释放激素基因的cDNA克隆及表达分析

垂体腺苷酸环化酶激活多肽 (PACAP)和生长激素释放激素 (GHRH)均属于血管活性肠肽家族成员 ,且两者前体基因在脊椎动物的鸟类、两栖类、鱼类中由同一基因编码 ,而哺乳动物是由两个不同基因编码。已有几例关于鱼类编码PACAP和GHRH基因克隆的报道 ,而关于重要海水养殖鱼类石斑鱼的PACAP和GHRH基因未见报道。克隆了PACAP GHRH前体cDNA序列 ,该前体有两种剪接方式 ,包括一个长序列和一个短序列 ,其中长序列编码PACAP和GHRH ,短序列缺失 10 5个碱基 ,仅编码PACAP而缺失编码GHRH的外显子区 ,同样情况在虹鳟和沟鲶中也有报道。通过半定量RT PCR方法对石斑鱼PACAP GHRH前体mRNA在胚胎发育和发育早期以及各部位的表达情况进行了分析。胚胎发育分析结果表明 ,从神经胚期开始 ,PACAP GHRH前体mRNA大量表达 ,提示该蛋白质在神经发育或神经营养方面具有重要作用。PACAP GHRH前体基因在中枢系统的表达量远高于外周组织。在鱼类的眼和鳃发现PACAP GHRH前体分布。

Sequence and Expression of a cDNA Encoding Both Pituitary Adenylate Cyclase Activating Polypeptide and Growth Hormone-releasing Hormone in Grouper (Epinephelus coioides)

Both pituitary adenylate cyclase activating polypeptide(PACAP) and growth hormone-releasing hormone (GHRH) belong to the vasoactive intestinal polypeptide-glucagon-secretin family. They are encoded on the same gene in vertebrates (birds, amphibian, fish). Although the gene encoding PACAP and GHRH has been cloned in other fish species, characterization of this gene in the commercially important grouper ( Epinephelus coioides ) has not been previously reported. In this study, the GHRH/PACAP cDNA was cloned from grouper hypothalamic tissue. Two cDNA variants of the GHRH/PACAP precursor gene were identified as a result of alternative splicing, a long form encoding both PACAP and GHRH and a short form encoding only PACAP. Both the long and the short forms of the GHRH/PACAP precursor cDNA were identified in grouper 22 tissues as well as expression in embryos and larvae by semi-quantitative RT-PCR detection. PACAP / GHRH precursor mRNA was expressed at a high level in the central nervous system than in several peripheral tissues. The data presented here provide the report of PACAP/GHRH mRNA expression in eye and gill tissues in fish. PACAP/GHRH mRNA was expressed in grouper embryo and all larval stages examined and was expressed at a high level starting on neurula stage onwards. The result suggests that PACAP/GHRH had an important role in the development of embryos and larvae, especially in neurula appearance stage.