The Fuction Of Retinoblastoma-binding Protein 4 Gene In Embryo Diapause Termination Of Brine Shrimp,Artemia Sinica

In present study,The full-length cDNA of As-rbbp4 from A.sinica(GenBank accession number: AKG51637.1)was obtained by using 3? and 5? RACE technology.It was found to contain 1411 bp,with a 1281 bp ORF and 63-bp 5?-UTR and a 67-bp 3?-UTR.Protein domains of As-RBBP4 were predicted by using the SMART online software,and results showed that it contains a WD40 superfamily domain with seven WD40 repeat sequences,which is a major feature of RBBP4.Analysis of As-RBBP4 by the TMHMM Server 2.0indicated that it is not a transmembrane protein.The subcellular localization of As-RBBP4 was predicted by the PSORTII program,which found that it may reside in the nucleus.Prediction by the PSORTII program showed that As-RBBP4 contains 24 phosphorylation sites(score > 0.5): including serine loci 16,threonine site 3 and tyrosine site 5.A signal peptide was not found,suggesting that As-RBBP4 is not a secretory protein.The hydrophobicity analysis of As-RBBP4 indicated that the protein is hydrophilic.Multiple sequence alignment analysis of the As-RBBP4 protein indicated that it is a conserved protein,which was also shown by protein sequence domain analysis,especially in the WD40 domain.Phylogenetic analysis revealed that the RBBP4 protein is highly conserved in A.sinica and other species,such as vertebrates.A.sinica has a relatively low classification status.The results of in situ hybridization showed that there is no specific of the gene in tissues or organs.The results of Real-time qPCR and western blot respectively from the expression of gene and protein level showed that reached a notably high level at 10 h,then began the obvious downward trend.There were obviously upregulation trend under low temperature stimulation and high salinity stress.As-E2F1 and As-CyclinE also presented a similar trend of As-RBBP4 in western blot.The expression pattern of RBBP4 in early embryonic development process of A.sinica was analyzed,experimental results showed RBBP4 played an important role in diapause termination and cell cycle regulation.