The Functional Characterization Of As-kip2 During The Restart Process Of Diapause Embryonic In Artemia Sinica

Artemia Sinica(Arthropoda, Crustacea, Anistraca, Artemiidae, Artemia). Is a small aquatic crustacean that is distributed widely in the inlang hypersmotic salt lakes and coastal lagoons in China. Artemia has an unusual diapause process under conditions of high salinity and low temperature stress. And in the right conditions, Artimia Sinica can restart growth. But the molecular mechanism of the restart process of diapause embryonic has not been understood.Kip2, a member of the Cip / Kip family, can also be called p57Kip2, CDKI1 C, belonging to cyclin-dependent kinase inhibitor(CDKI). It’s a negative regulator of cell cycle. CDKI can inhibit activity through competitively binding to cyclin-dependent kinase(CDK), cell cycle arrest in G1 phase, in order to achieve the regulation of the cell cycle.In this study, Artimia sinica were used as experimental materials. And the 1023 bp full-length cDNA of Kip2 from Artemia sinica(As-Kip2) was obtained. Molecular characterization revealed that the sequence includes an 492 bp open reading frame encoding a predicted protein of 163 amino acids. The predicted protein contains CDI domain. As-KIP2 is a protein is about 18.7kDa with an isoelectric point of 9.45. The predicted protein is a hydrophobic protein, has 15 phosphorylation sites, no signal peptide, no transmembrane domain. The kinship was found farther on evolution with other species by analysis of the evolutionary tree. Real-time quantitative PCR was used to determine the expression level of As-Kip2 during embryonic development and low temperatures challenge and high salty challenge. The result shows the expression increased gradually with the development of embryonic. And the expression level shows higher under low temperatures and high salt conditions. In situ hybridization was used to determine the expression site of As-Kip2 in different development period, and found that As-Kip2 was expressed in various parts of Artemia sinica. Western blotting was used to determine protein production of As-Kip2 during embryonic development, low temperatures challenge and high salty challenge. It’s shows that with the development of Artemia sinica, the temperature decreases, the salinity gradually increased, As-KIP2 protein production gradually increased, which similar to the results of Real-time qPCR. 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 As-Kip2 in vertebrates.