The Potential Role Of APC/C-CdC20 During The Diapauses Embryonic Restarting Process Of Artemia Sinica

Artemia sinica is a small crustacean widely living in high-salinity water body with strong resistance, Artemia enter diapause stage in response to unfavourable environments and form a special diapause cysts to arrest development temporarily. Due to the characteristic, Artemia become a model for the study of biological experiments.Ubiquitination through a series of enzyme, E1, E2, E3 catalysis, ultimately degrade the substrates. The anaphase promoting complex, cyclosome, APC/C is a representative E3 ubiquitin ligase and its targets specific mitotic regulators are degraded by the 26 S proteasome. CDC20 is one of the two cofactors of the APC/C, it can bind and activate APC complex at a particular point in time and plays an important role in the process of guiding ubiquitin protein degradation of the cell cycle and ensuring normal chromosome separation. As most cell cycle-relate proteins degraded through the ubiquitination, CDC20 plays a key role in presses of the cell cycle regulation. However, the role of As-cdc20 in the regulation of the cell cycle during the diapause embryonic restarting process still remains unknown.In this study, a 1842 bp full-length cDNA of cdc20 from A. sinica(As-cdc20) was cloned for the first time, with an open reading frame(ORF) of 1512 bp, 5’UTR of 50 bp and 3’UTR of 280 bp, it encodes a 503 amino acids, 55.5 kDa protein. Multiple sequence alignment analysis showed that CDC20 was a conserved protein, especially in the WD40 domain. Bioinformatics analysis showed As-CDC20 primarily localized in the nucleus, as a non-transmembrane hydrophilic non-secreted protein, contains 30 predicted phosphorylation sites. Results of in situ hybridization showed that there is no tissue or organ specificity on the expression locations of As-cdc20. The expression levels of As-CDC20 were analysed by Real-time qPCR and Western blotting, the results showed that the highest expression level was at the 5 h stage of embryonic development, and gradually reduced with the development of Artemia, meanwhile, the expression level of As-CDC20 was also reduced by stresses such as low temperature and high salinity.The experimental results describes the role of CDC20 in the regulation of the cell cycle in Artemia sinica early embryonic developmental stages, it suggested that As-CDC20 is a crucial protein in the diapauses embryo restarting process of A. sinica.The results have a significance meaning for further study of the mechanism of formation and the termination in the diapause process of A. sinica and the datas presented here provide the basis for further studies of CDC20 in vertebrates.