Cloning and Expression Analysis of Cdc25 Gene of <i>Sipunculus nudus</i> in Oocytes

Abstract

Cell division cycle 25 ( Cdc25) is an important dual specificity phosphatase, which plays an important role in regulating the process of oocytes meiosis and embryo development. In our study, the full-length cDNA of Cdc25 from S. nudus (Sn-Cdc25) was cloned by 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 which 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, Rhodanese-like domain, and the active site sequence HCX5R which 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 their active site is highly conservative. The 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: invertebrate and vertebrate. qRT-PCR results show that the expression of Sn-Cdc25, with two peaks, is significantly different in different developmental stages of oocytes. The increased expression of Sn-Cdc25 may be related to the process of Cdc25 promoting DNA replication from the the early stage of yolk formation to the late stage of vigorous yolk synthesis (O1-O3). 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 the optimization of artificial breeding techniques.