光裸星虫Cdc25基因的克隆及在卵母细胞中的表达分析

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

细胞分裂周期蛋白25 (cell division cycle 25, Cdc25)是一种重要的双特异性磷酸酶,对卵母细胞减数分裂进程和胚胎发育具有重要的调控作用。本研究利用RACE技术克隆获得了光裸星虫(Sipunculus nudus) Cdc25(Sn-Cdc25)cDNA全长。Sn-Cdc25全长为4 130 bp,其中3’UTR为1 849 bp,5’UTR为427 bp,开放阅读框为1 854 bp,编码617个氨基酸。序列分析显示,Sn-Cdc25蛋白分子量为69.58 kD,具有M相诱导磷酸酶结构域(M-phase inducer phosphatase domain)和硫氰酸酶同源结构域(rhodanese-like domain)两个典型的Cdc25蛋白结构域,以及能催化去磷酸化过程的活性位点序列HCX_(5)R。多序列比对发现Cdc25同源蛋白的C端同源性较N端的高。三级结构预测表明Cdc25同源蛋白及其活性位点的三级结构构象高度相似。motif分析中共发现5个motifs,其中motif 1和motif 2分别为Paxillin LD基序和MYND结构域结合基序。系统进化树分析显示,无脊椎动物和脊椎动物的Cdc25分别聚为两大支。RT-qPCR结果显示,在不同发育时期卵母细胞中,Sn-Cdc25的表达差异显著,具有两个峰值,从卵黄形成初期至卵黄旺盛合成后期(O1~O3)Sn-Cdc25表达量上升可能与Cdc25促进DNA的复制过程相关;而从体腔液中进入到肾管后(O4~O5),表达量的迅速上升可能有利于成熟促进因子(maturation promoting factor, MPF)的活化。研究结果为深入认识星虫动物卵母细胞发育调控机制和优化人工繁育技术积累基础资料。

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 this study, the full-length cDNA of Cdc25 from Sipunculus nudus(Sn-Cdc25) was cloned by using RACE technology. The results showed that Sn-Cdc25 was 4 130 bp in length, including 3’ UTR 1 849 bp and 5’ UTR 427 bp. The open reading frame(ORF) was 1 854 bp which encoded 617 amino acids. Sequence analysis showed that the molecular weight of Sn-Cdc25 protein was69.58 kDa, with two typical Cdc25 protein domains: M-phase inducer phosphatase domain and rhodanese-like domain, and the active site sequence HCX_(5)R which can catalyze the dephosphorylation process. Multi-sequence alignment found that the C-terminal homology was higher than N-terminal. The tertiary structure prediction showed that the spatial conformation of Cdc25 homologous protein and their active site were highly conservative.The total of 5 motif s were found in motif analysis, of which motif 1 and motif 2 were Paxillin LD motif and MYND domain binding motif, respectively. Phylogenetic tree analysis showed that Cdc25 was clustered into two branches: invertebrates and vertebrates. RT-qPCR results showed that the expression of Sn-Cdc25, with two peaks,was 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(O4~O5),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.