Cloning and characterization of DrSpin-1 gene in zebrafish (Danio Rerio)

Spin protein family, with the conserved Spin/Ssty motif, play important role during gametogenesis and early embryo development. So far, no Spin genes have been identified in zebrafish,Danio rerio. In this study, to isolate the full-length cDNA of Spin gene from zebrafish, we designed two degenerate primers according to the conserved sequences of Spin protein family. Two 260 bp DNA fragments of DrSpin-1 and DrSpin-2 were isolated from SMART cDNA library of zebrafish mature eggs. The sequence homologous analysis indicated the identity of amino acid sequences between DrSpin-1 protein and CagSpin protein got up to 81%, and DrSpin-2 protein showed greater sequence identity with OlSpinB and OlSpinC. Further, two specific primers were designed according to the 260 bp DNA sequence of DrSpin-1 for RACE polymerase chain reaction, and the full-length cDNA sequences of DrSpin-1 was isolated from the SMART cDNA library referred above. Sequence analysis revealed DrSpin-1 cDNA contained 1082 base pairs with a 771 base pairs open reading frame encoding 257 amino acids and including three conserved Spin/Ssty motives and eight possible phosphorylation sites. These data confirmed the cloned DrSpin-1 gene was one of Spin gene family. Furthermore, the multiple alignments analysis of DrSpin-1 protein sequence and Spin protein sequences in other fishes by CLUSTRA W indicated that DrSpin-1 protein had the highest homology to CagSpin protein in gibel carp. Therefore, we suggested DrSpin-1 in zebrafish might have the similar expression pattern to CagSpin, and might play important roles in regulation during oogenesis and fertilization. Our studies provide the clue to further functional research of Spin in zefrafish.