Cloning,Expression Pattern And Potential Role Of SKP1 In Diapause Termination Of Embryo From Artemia Sinica

Artemia sinica is an important bait organism and experimental animal in crustaceans.When Artemia encounters a harsh environment,their growth and development will stop and form diapause embryo to avoid the bad environment.When the living conditions are suitable,the development of the embryo is restarted,and this process must be accompanied by the degradation of proteins related to embryo diapause.Protein degradation includes multiple pathways such as ubiquitination,lysosomes,and Caspase.Among them,the degradation pathway of ubiquitinated proteases is the most widely studied protein degradation pathway.Ubiquitin-activating enzyme E1 uses the energy released by ATP hydrolysis to activate the ubiquitin molecule Ub,and then transfer the activated ubiquitin molecule to the binding enzyme E2,followed by the E3 ligase to connect the ubiquitin-binding E2 to the target protein,and finally by hydrolysis The target protein completes the ubiquitination process.In this research,we studied the key skeleton protein SKP1(S-phase kinase-associated-protein 1)of SCF(Skp1-Cul1-F-box)in the E3 ubiquitin ligase complex during the early embryonic development of Artemia.SKP1 can participate in stabilizing the F-Box conformation and mediate protein degradation,and plays an important role in the early development,But at present,the regulation and function of SKP1 in Artemia diapause is still unclear.In this research,the full-length c DNA of As-skp1 gene of A.sinica was cloned for the first time,with the gene sequence number: QGN75283.1,and bioinformatics analysis was carried out.The real-time quantitative PCR method was used to detect the m RNA expression level and expression pattern of As-skp1 in various stages of the development of A.sinica embryos and under the conditions of temperature stress and salinity stress.As-SKP1,As-SKP2,As-CDK2 and As-CUL1 protein expression patterns and levels during the development of Artemia embryos,according to immunofluorescence technology,the expression site of As-SKP1 in A.sinica was detected.The role and function of As-skp1 were analyzed by si RNA technology.The experimental results show that As-skp1 is 659 bp in length and encodes 163 amino acids.As-skp1 showed a high-to-low expression trend during the development of Artemia embryos,and its relative expression level was highest at 15 h after development.The protein expression level is basically consistent with the change of gene expression trend.As-SKP1 immunofluorescence experiments showed that there is no specificity of organs and tissues.si RNA experiments show that knockdown ofAs-skp1 leads to an increase in deformity rate.The following conclusions were obtained through experiments: As-skp1 is a gene related to cell cycle regulation.The high expression before Artemia embryo development to 15 h is due to the large amount of material reserves required for cell proliferation and cell division after embryo start-up,and embryo development to 15 The expression level was down-regulated after h,which proved that the embryonic cell-related activities decreased,the cells began to differentiate,and related genes were down-regulated,and finally maintained at the basic level.The results of real-time fluorescence quantitative q PCR and Western blot all confirmed this result.After As-skp1 is knocked down,there will be stunting and a large number of abnormal embryo.Therefore,SKP1 is important for the restart of Artemia embryo and diapause release.The research on SKP1 will be helpful to understand the restart mechanism of Artemia diapause embryo,analysis of ubiquitination pathways of protein degradation and develop the Artemia resources has potential application value.