Molecular cloning and sequence analysis of full-length growth hormone cDNAs from six important economic fishes.

In this study,the full-length cDNAs of GH (Growth Hormone) gene was isolated from six important economic fishes, Siniperca kneri, Epinephelus coioides, Monopterus albus, Silurus asotus, Misgurnus anguillicaudatus and Carassius auratus gibelio Bloch. It is the first time to clone these GH sequences except E. coioides GH. The lengths of the above cDNAs are as follows: 953 bp, 1 023 bp, 825 bp, 1 082 bp, 1 154 bp and 1 180 bp. Each sequence includes an ORF of about 600 bp which encodes a protein of about 200 amino acid: S. kneri, E. coioides and M. albus GHs of 204 amino acid, S. asotus GH of 200 amino acid, M. anguillicaudatus and C. auratus gibelio GHs of 210 amino acid. Then detailed sequence analysis of the six GHs with many other fish sequences was performed. The six sequences all showed high homology to other sequences, especially to sequences within the same order, and many conserved residues were identified, most localized in five domains. The phylogenetic trees (MP and NJ) of many fish GH ORF sequences (including the new six) with Amia calva as outgroup were generally resolved and largely congruent with the morphology-based tree though some incongruities were observed, suggesting GH ORF should be paid more attention to in teleostean phylogeny.

[Polymorphism in intron 2 of crucian carp GH I gene].

High length and nucleotide polymorphisms in intron2 of GH I gene were detected in 162 individuals,which were from seven wild crucian carp colonies, two goldfish colonies and one Fangzheng crucian carp colony. Using denaturating polyareylamide gel electrophoresis (DPAGE) and single-strand conformation polymorphism (SSCP), seven length variants and 15 haplotypes were identified in these fishes. The length types and haplotypes diversity was 4.32% and 9.26%, respectively. Sequence analysis of the 15 haplotypes indicated the following results: (1) The size of intron2 varied from 243 to 263 bp. In the 15 haplotypes,the average percentages of the four bases (A,T,G and C) were 34.13%, 37.36%, 15.13% and 13.38%, respectively; the frequency of G + C(28.51%) was much lower than that of A + T (71.49%). The GT/AG domain was found in exon-intron junctions,which was the 5' and 3' splice donor and acceptor sites in higher eukaryotic gene introns. The similarity sequence of GTAAGTA was located on the junction between exon2 and intron2. And there existed a richer pyrimidine region (TTTGCCTTTTGTTATC) near the 3' end of intron2. (2) The seven length variants (A, B, C, D, E, F and G) were determined to be 189, 196, 204, 205, 206, 207 and 209 bp, respectively. The polymorphism resulted from the variable repeat number of T (N = 0, 8, 9, 10, 11 and 13) and the difference in one or two motifs deletions of TGAAAAC, TT and GAGTG. (3) Compared the sequences of the 15 haplotypes, 17 substitution sites were observed, of which two were of transversion sites and 15 were of transition sites. Obviously, the transition mutations (88.24%) were more frequent than transversion mutations (11.76%). Analysis of the distributions of the length types, haplotypes and composite genotypes suggested that genetic diversity was varied in different colonies. In the goldfish colonies, only one length type (A), two haplotypes (A1 and A2) and one composite genotype (A1A2) were observed. Two length types (C and D), four haplotypes (C1, C2, D2 and D5) and one composite genotype (C1C2D2D5) presented in the Fangzheng crucian carp colonies. The highest level of genetic diversity was exhibited in the seven wild crucian carp colonies: seven length types (A, B, C, D, E, F and G), 14 haplotypes (A1, A2, A3, B, C1, C2, D1, D2, D3, D4, E1, E2, F and G) and 14 composite genotypes (A1A2A3, A1A2A3B, A1C1C2D1D2D3, A1C1C2D2, A1C1C2D2D3, A1C1C2D3E2, BC1C2D2, BC1C2D3D4, C1C2D2, C1C2D2D3, C1C2D3D4, C1C2D3D4F, C1C2D4, D2E1G) were shared by the seven wild colonies. The numbers of observed length types, haplotypes and genotypes within the wild colonies ranged from 3 to 6, 6 to 10 and 2 to 6, respectively. Whether the length and nucleotide polymorphisms in the intron2 of crucian carp GH I gene were associated with gene expression and gene regulation remained unsolved and required further investigations.