[Cloning of hemoglobin alpha-chain cDNA sequences from five fishes in cypriniformes]

Oxygen content in water is one of the limit factors to the fish growing. The tolerance of fish to low oxygen content depends on the affinity of hemoglobin to oxygen. The tempt of transgenic fish study is to transfer hemoglobin gene from the fish that tolerates to low oxygen content in water to the fish sensitive to low oxygen condition. Therefore, using hemoglobin gene to breed new varieties may become a main trend in the near future. To explore the underline mechanisms for the variation of hemoglobin property, we have cloned hemoglobin-alpha-chain from five species of Cypriniformes fishes. A pair of consensus degenerate primes were designed basing on N-terminal and C-terminal conservative amino acid of alpha-chain. Using RT-PCR method, alpha-globin gene was amplified and cloned from total RNA which was extracted from blood of five fishes which are widely distribution in China (Cyprinus carpio, Ctenopharyngodon idellus, Carassius auratus, Misgurnus anguillicaudatus, Paramisgurnus dabryanus). Sequence comparison with the existing hemoglobin alpha-chain confirmed that these cDNA fragments were alpha-globin gene of five fishes. The accession numbers in GenBank are AF528156, AF528157, AF528197, AF528198, AF528199 respectively. The cDNA which coding amino acid are all 429 bp. Compared with the amino acid sequences of five fishes, it can be concluded that in five fishes the highest similarity of two fishes which are Paramisgurnus dabryanus and Misgurnus anguillicaudatus is 99.41%, whereas the lowest similarity of two fishes which are Ctenopharyngodon idellus and Paramisgurnus dabryanus is 83.92%. In addition, the sequence similarity and phylogenetic relationship were compared with Within-group method. The result shows that there is much consistent between similarity and phylogenetic relationships from morphological of those near relation species and much connection between similarity and environment of those distant relation species.