RACE法分离团头鲂生长抑素全长cDNA及其序列测定

生长抑素具有抑制脑垂体GH释放的作用，是调控鱼类生长的主要激素之一。本研究采用RT和RACE(rapid amplification of cDNA ends)法，分离和测定了团头鲂脑中生长抑素PSSI cDNA的全长核苷酸序列，并对该基因进行了结构和系统进化分析。3’RACE扩增得到700bp左右的片段，5’RACE分离得到500bp左右的片段，把3’片段与5’片段拼接得到全长cDNA。cDNA全长735bp不含poly(A)]，5’端非翻译区有100nt。3’端290bp不包含poly(A)]，阅读框(open reading frame，ORF)345bp。该序列与金鱼PSSI cDNA序列同源性为90％，主要差异在5’端非翻译区。团头鲂生长抑素mRNA阅读框编码114个氨基酸，包括一些酶切位点，产生26个氨基酸的大分子态生长抑素，进一步加工成与人等结构相似的14个氨基酸的生长抑素；团头鲂生长抑素前体氨基酸序列与金鱼、虹鳟、鲶鱼、鲅鱇、蛙、牛、鼠、鸡、猴、人等的比较发现，它与金鱼的同源性最高达95％，与鮟鱇最低50％，与人68％。这说明生长抑素基因在长期的进化中相当保守，同时，也因为鱼类生活环境多样导致基因变异较大。

Isolation and sequence somatostatin cDNA of Megalobrama pellegrini

Somatostatin is one of important hormones in fish growth regulation, which was characterized as a physiological inhibitor of GH secretion in pituitary. In this study, a two-phase rapid amplification of cDNA ends (RACE)-PCR was used for the isolation of the full length cDNA of propresomatostatin I from brain of blunt bream Megalobrama pellegrini. The structure and phylogenesis of this gene were also analyzed. A fragment about 700bp was amplified in 3' RACE-PCR, and sequenced. Based upon this sequence a second gene-specific primer for 5' RACE-PCR was designed which yielded an about 500bp fragment. Sequence analysis revealed a 735bp cDNA containing the 100nt 5'-untranslated region, 290bp 3'-untranslated regions and 345bp open reading frame which encode a preprosomatostatin of 114 amino acid. The deduced 114-amino acid PPSS contains a number of putative processing sites, potentially yielding a 26-amino acid peptide that could be processed further to a 14-amino acid peptide identical in structure to mammalian SS-14. Sequence analysis revealed the identity rate of cDNA of propresomatostatin I of blunt bream and goldfish is 90 % . Alignment score between deduced amino acid sequences of propresomatostatin I from M. pellegrini and goldfish , rainbow trout, catfish, anglerfish, frog, cow, rat, chicken, monkey, human being is 95% ,78% ,74% ,50% ,70% ,67% ,65% ,75% , 68% , and 68% respectively. These results indicate the somatostatin gene is conservative in the progress of evolution, meanwhile, it is also the cause of the variation of dwelling environment of fish that induces the great gene differences.