斑马鱼DrSpin-1基因的克隆和特征分析

CLONING AND CHARACTERIZATION OF DRSPIN-1 GENE IN ZEBRAFISH (DANIO RERIO)

Spin蛋白家族是具有Spin/Ssty保守结构域并在配子发生过程中发挥关键作用的一类分子。研究利用简并引物PCR,从斑马鱼成熟卵母细胞SMART cDNA文库中筛选到260 bp的DrSpin-1和DrSpin-2部分序列,经序列同源性比对,斑马鱼DrSpin-1的部分氨基酸序列与银鲫CagSpin一致性高达81%。利用RACEPCR从该cDNA文库中获得斑马鱼DrSpin-1的全长cDNA序列。序列分析表明,DrSpin-1全长cDNA为1082 bp,开放阅读框771 bp,编码257个氨基酸,具有三个Spin/Ssty保守域,8个可能的磷酸化位点,初步确定斑马鱼DrSpin-1是Spin基因家族成员。斑马鱼DrSpin-1蛋白与已报道的鱼类Spin蛋白多重序列比对表明,DrSpin-1蛋白与银鲫CagSpin蛋白同源性最高。可以推测克隆得到的斑马鱼DrSpin-1与已知功能的银鲫CagSpin具有相近的表达谱和生物学功能,可能在配子发生和受精过程中发挥重要作用。

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.