CLONING AND SEQUENCE ANALYSIS OF INSULIN-LIKE GROWTH FACTOR-I GENE IN TOPMOUTH CULTER (CULTER ALBURNUS BASILEWSKY)

In order to study the effect of IGF-Ⅰ gene on growth traits of topmouth culter (Culter alburnus Basilewsky), IGF-Ⅰ gene was cloned from the genomic DNA of topmouth culter in this study. The gene spanned 14567 bp and comprised five exons and five introns. The five exons of IGF-Ⅰ were 298, 160, 182, 36, and 1360 bp long, respectively. The hypothetical open reading frame of the IGF-Ⅰ cDNA precursor was 486 bp, encoding a putative protein of 161 amino acids. The precursor peptide included a signal peptide (44 amino acids), a mature peptide (70 amino acids), and an E peptide (47 amino acids). The mature peptide comprised four regions: B, C, A, and D. The A and B domains were conserved with six cysteine residues in these two regions to form three disulfide bonds. The B region contained a conserved IGF-Ⅰ receptor recognition sequence (Phe^B23-Tyr^B24-Phe^B25). Analysis of the E peptide showed that IGF-Ⅰ of the topmouth culter was an Ea-2 type. The IGF-Ⅰ amino acid sequence of the topmouth culter had high sequence identity with IGF-Ⅰ proteins from Cyprinidae fish, ranging from 94 to 100%. Phylogenetic analysis of IGF-Ⅰ amino acid sequences showed that IGF-Ⅰ of the topmouth culter does not cluster with the fish from subfamily Cultrinae. IGF-Ⅰ is expressed in all 10 tested tissues with the highest mRNA level in liver, modest level in spleen, heart, testis, and brain, and low level in kidney, gill, stomach, and ovary. The lengths of the four introns of topmouth culter IGF-Ⅰ were 1170, 9364, 251, and 1746 bp, respectively. The variation in the sequence of the introns among species was greater than that of the exons with the highest variation in the third intron. Six microsatellite loci were found in the introns of IGF-Ⅰ with (GATG)₅AATAT (ATAG)₁₁ in intron 1, and (CT)₈, (TTA)₅, (AC)₁₃, (TG)₁₂, and (ATT)₅ in intron 2. Four of 6 microsatellite loci were polymorphic, and the genotypes of the four microsatellite loci had no significant correlation with the growth traits of 120 topmouth culter cultured in the same pond (P>0.05). These results provide molecular basis for studying the expression pattern, function and transcriptional regulation of theIGF-Ⅰ gene.