Amplification, cloning, and sequence comparison of the growth hormone gene for carp (Cyprinus carpio) by the polymerase chain reaction

Abstract

Total RNA was isolated from carp pituitary gland. The first strand cDNA was synthesized using oligo(dT) 12-18 as a primer, the total RNA as a template, and AMV reverse transcriptase. Next the polymerase chain reaction (PCR) was performed using the first strand cDNA as a template, the synthetic 29 oligonucleotides as primer and Taq DNA polymerase (94 degrees C, 60 s; 55 degrees C, 30 s; 72 degrees C, 50 s; 35 cycles). After PCR amplification, the products were cloned into an E. coli expression vector (PBluescript II KS+/-). The result of the sequence analysis and the restriction map shows that an open reading frame of the carp growth hormone gene contains 630 base pairs which code for a polypeptide of 210 amino acids including 22 amino acids of the signal peptide and 188 amino acids of the nature growth hormone. Nucleotide sequence and amino acid sequence of our carp growth hormone gene are the same as Koren's carp GH cDNA in the coded region. Compared with Chao's carp GH cDNA, the homology of nucleotide sequence and amino acid sequence for our carp growth hormone gene is 95.6% and 96.7%, respectively, in the coded region.