Cloning of CyclinB gene from Sipunculus nudus and its expression analysis in oocytes

【Objective】 Exploring the role of CyclinB gene in the development and maturation of Sipunculus nudus oocytes to provide a theoretical basis for further understanding of the molecular mechanism of S.nudus oocyte development.【Method】 RACE technology was used to clone the full length of S. nudus CyclinB(Sn-CyclinB) cDNA and the bioinformatics analysis was performed by some online softwares like ProtParam, DNAMAN 9.0, COBALT, SOPMA, Phyer2, Pfam, and BLAST. The real-time fluorescence quantitative PCR technology was used to analyze the expression pattern of Sn-CyclinB in different developmental stages of oocytes.【Result】 The full-length cDNA of Sn-CyclinB was 2468 bp with 141 bp 5'-untranslated region(5'-UTR), 1097 bp 3'-untranslated region(3'-UTR), and 1230 bp open reading frame(ORF). It encoded 409 amino acids and contained three cytoplasmic polyadenylation elements(CPE), a translation control element(TCE) and a K-box translation regulation element in the 3'-UTR. The theoretical molecular weight and isoelectric point(pI) of Sn-CyclinB protein were 46.304 kD and 8.68 respectively and it had a signature sequence(FLRRxSK) and Cyclin degradation box(RxALGxIxN) which was a hydrophilic protein. Sn-CyclinB protein also contained a cyclin box and the secondary structure of protein, alpha-helix, extension chain, beta-corner, and random curl accounted for 56.72%, 3.91%, 0.73%, and 38.63% respectively. It suggested that the C-terminus of CyclinB homologous protein was more conservative than N-terminus. Compared with other species, the position of the Cyclin degradation box(RALGTISN) had moved forward by 13 amino acids and the tertiary structure of Sn-CyclinB protein was highly similar to those of Capitella teleta and Patella vulgata. Phylogenetic tree analysis, based on the similarity of amino acid sequences of CyclinB, showed that CyclinB clustered into invertebrates and vertebrates branches among which S.nudus, C. teleta and Helobdella triserialis were clustered into the invertebrate branch. Sn-CyclinB was expressed in different development stages of oocytes and the trend was bimodal. The higher expression was in the late yolk vigorous synthesis period(O3) and the nephridioduct development period(O5). 【Conclusion】 Sn-CyclinB protein has CyclinB feature sequences and its 3'-UTR has many kinds of regulation elements which relating to translated regulation. Sn-CyclinB protein may be involved in the meiosis transition of G₁/S and G₂/M phases of the S. nudus oocytes and it will play a crucial role in promoting the orderly progress of oocyte meiosis.