Molecular Cloning, Characterization And Expression Of Insulin-like Growth Factors From Mandarin Fish Siniperca Chuatsi

In this study, the complete cDNA sequences of insulin-like growth factor-Ⅰand -Ⅱof Siniperca chuatsi liver were isolated by means of RT-PCR, rapid amplification of cDNA ends (RACE) and other cloning techniques, their structure characteristics were also analyzed. Using real-time quantitative RT-PCR, their expression level in different adult tissues and larval development stages (days after hatching of 0, 2, 4, 6, 8, 10, 12, 14, 16, 19) were also detected. Results showed that: (1) The complete cDNA of insulin-like growth factor-Ⅰwas 1784 bp, it contained a 233 bp 5′-untranslated region, 990 bp 3′-untranslated region and 561 bp open reading frame (ORF), which encoded 186 amino acids with a putative signal peptide of 44 amino acids, a mature peptides of 68 amino acids, and a E peptides of 74 amino acids, the mature peptides includes B, C, A and D domains, E peptides analysis showed that S. chuatsi IGF-Ⅰbelong to Ea-4 type. The similarity of S. chuatsi IGF-Ⅰamino acid sequence was 81%-99% with that of other vertebrate. The complete cDNA of insulin-like growth factor-Ⅱwas 1643bp, containing 103 bp 5′-untranslated region, 892 bp 3′-untranslated region and 648 bp open reading frame (ORF), which encoded 215 amino acids with a signal peptides of 47 amino acids, a mature peptides of 70 amino acids and a E peptides of 98 amino acids, the mature peptides includes B, C, A and D domains. The similarity of S. chuatsi IGF-Ⅱamino acid sequence was 81%-98% with that of other vertebrate, but only 62.5% with that of S. chuatsi IGF-Ⅰ. (2) IGF-ⅠmRNA was detected the highest expression in the liver, and high expression in kidney, brain, hindgut, skin, gonad, but low expression in stomach, spleen, foregut, gill, heart, muscle; it was continuously expressed during the early development and the expression level was gradually increased. IGF-ⅡmRNA was detected the highest expression in the brain, it also high expressed in muscle, heart, stomach, gill and kidney, but low in spleen, foregut, hindgut, gonad and liver; it was continuously expressed during the early development, the highest expression level was found at the first hatching day, later the expression was reduced slightly. These results may build the molecular base for further study on growth and development regulation of IGFs.