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.
Highlights
The course of phosphorylation and non-phosphorylation of protein is a significant molecular mechanism that regulating function protein activity (Hunter, 1995)
The full-length cDNA of Cell division cycle 25 (Cdc25) was cloned from S. nudus (Sn-Cdc25) using RACE technology, and we analyzed its sequence feature and relative expression of Sn-Cdc25 in oocytes at different developmental stages to explore the function of Sn-Cdc25 in the oocytes development process
Protein phosphatase is divided into serine/threonine phosphatases, tyrosine phosphatase and dual specificity phosphatase (DSP)
Summary
1.1 Gene sequence analysis of Sn-Cdc The gene sequence of Sn-Cdc, which has the active site sequence HCX5R, is 4 130 bp in length, including 3'UTR 1 849 bp and 5'UTR 427 bp and the open reading frame (ORF) is 1 854 bp which encodes 617 amino acids (Figure 1). The prediction of the secondary structure shows that the random coil of Sn-Cdc protein is the highest (66.61%), and the alpha helix is the second highest (24.80%) that mostly exist C-terminal (Figure 2). The α-helix region (aa: 549-556) and the active site of Cdc are separated by two amino acid and they maybe perform the biological function mutually. 1.3 Tertiary structure analysis of Sn-Cdc protein Forecast the protein tertiary structure of S. nudus, Lingula anatine (XP_013383189.1), Octopus vulgaris (XP_029639960.1), Mizuhopecten yessoensis (XP_021352882.1). The result finds that the spatial conformation of Cdc homologous protein and their active site is highly conservative, and the invagination of the active site is shallow (Figure 3). The result shows that the homology and conservation of Cdc protein are higher in C-terminal relatively. In N-terminal, the homology is lower and the variation extent is larger relatively (Figure 4)
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