Cloning and Expression Analysis of Cdc25 Gene of Sipunculus nudus in Oocytes

Abstract

Cell division cycle 25 (Cdc25) is an important dual specificity phosphatase, which plays an important role in regulating the process of oocyte meiosis and embryo development. In this study, the full-length cDNA of Sn-Cdc25 was cloned from S. nudus using RACE technology. The results show that Sn-Cdc25 is 4 130 bp in length, including 3′ UTR 1 849 bp and 5′ UTR 427 bp. The Open reading frame (ORF) is 1 854 bp and encodes 617 amino acids. Sequence analysis shows that the molecular weight of Sn-Cdc25 protein is 69.58 kD, with two typical Cdc25 protein domains: M-phase inducer phosphatase domain and Rhodanese-like domain, and the active site sequence HCX 5 R that can catalyze the dephosphorylation process. Multi-sequence alignment finds that the C-terminal homology is higher than the N-terminal. The tertiary structure prediction shows that the spatial conformation of Cdc25 homologous protein and its active site are highly conservative. A total of 5 Motifs are found in Motif analysis, of which Motif 1 and Motif 2 are Paxillin LD motif and MYND domain binding motif, respectively. Phylogenetic tree analysis shows that Cdc25 is clustered into two branches: invertebrates and vertebrates. RT-PCR results show that the expression of Sn-Cdc25 , with two peaks, is significantly different in different developmental stages of oocytes. The increase in the expression of Sn-Cdc25 from primary vitellogenic stage to the late of active vitellogenic stage (O1-O3) may be related to the process of Sn-Cdc25 promoting DNA replication. When the oocytes entering metanephridium from coelomic fluid, the rapid rise of Sn-Cdc25 expression may be beneficial to the activation of maturation promoting factor (MPF). The above results have accumulated basic data for further understanding of the developmental mechanism of Sipuncula oocytes and for optimization of artificial breeding techniques.