Cloning and study of adult-tissue-specific expression of Sox9 in Cyprinus carpio

The Sox9 gene is one of the important transcription factors in the development of many tissues and organs, particularly in sex determination and chondrogenesis. We amplified the genomic DNA of Cyprinus carpio using degenerate primers, and found that there were two versions of Sox9 in this species: Sox9a and Sox9b, that differ in having an intron of different length (704 bp and 616 bp, respectively) in the conserved HMG box region that codes for identical amino acid sequences. We used a two-phase rapid amplification of cDNA ends (RACE) for the isolation of full-length cDNA of Sox9b. Sequence analyses revealed a 2447-bp cDNA containing 233-bp 5' untranslated region, a 927-bp 3' untranslated region, including poly(A), and a 1287 bp open reading frame (ORF) encoding a protein of 428 amino acids. The HMG box of 79 amino acid motif was confirmed from positions 96-174. Sequence alignment showed that the identity of amino acids of Sox9 among ten animal species, including C. carpio, is 75%, indicating that the Sox9 gene is evolutionarily quite conserved. The expression level of Sox9b gene varied among several organs of adult C. carpio, with the level of expression being highest in the brain and testis.