大黄鱼(Larimichthys crocea)IGF-Ⅰ基因的克隆及序列分析

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF INSULIN-LIKE GROWTH FACTOR ⅠcDNA FROM LARIMICHTHYS CROCEA

采用RT-PCR和RACE技术克隆了大黄鱼胰岛素样生长因子-Ⅰ(IGF-Ⅰ)cDNA的全序列,序列全长2027bp,由57bp的5’非翻译区、1409bp的3’非翻译区和561bp的开放阅读框组成。序列分析表明,大黄鱼IGF-Ⅰ编码区包括信号肽、成熟肽(B、C、A、D)和E等6个区域的186个氨基酸,形成成熟肽时,信号肽和E区被切除,成熟肽分子量7.5kDa,等电点(pI)为7.76,成熟肽的B区和A区有6个保守的半胱氨酸残基,形成3对二硫键,起到了稳定IGF-Ⅰ三维结构的作用。与其它物种的IGF-Ⅰ比对后发现A、B区域比较保守,C、D区域保守性较差;E区域的分析结果表明,大黄鱼IGF-ⅠcDNA序列为Ea-4亚型。核苷酸同源性分析发现,大黄鱼IGF-ⅠcDNA与美国红鱼同源性最高,为99.47%;与斜带石斑鱼、金头鲷和褐牙鲆的同源性依次降低,分别为97.68%、97.50%和95.90%。

The insulin-like growth factor （IGF-Ⅰ） gene of Larimichthys crocea was cloned from liver by RT-PCR and RACE technique. All the length of the nucleotide sequence was 2027bp, including 5’UTR （57bp）, 3’UTR （1409bp） and ORF （561bp）. Nucleotide sequence analysis showed that ORF encoded 186 amino acids that was composed of signal peptide, mature peptide （B, C, A, D） and E domain. Signal peptide and E domain were cut off when the peptide was processed. The molecular weight of mature peptide was 7500Da and its pI was 7.76. There were three disulfide bonds formed by six cysteines in B and A domains. The disulfide bonds comparing the amino acid sequence of IGF-Ⅰof L. crocea with those of other species, the sequence conservation of A and B domains is high, while low in C and D domains. E domains in IGF-Ⅰ of L. crocea was belonged to IGF-ⅠEa-4 subtype. Analysis of nucleotide sequence showed that IGF-ⅠcDNA of L. crocea is similar to other fishes which shared sequence homology of 99.47% with Sciaenops ocellatus, 97.68% with Epinephelus coioides, 97.50% with Sparus aurata and 95.90% with Paralichthys olivaceus.