The Expression And Function Of As-NEDD4 In The Process Of Diapause Embryos Restart Of Artemia Sinica

Artemia sinica have a special mechanism of diapause,diapause eggs have extremely strong resistance and can be preserved in dry conditions for several years.When the external environment becomes better,the diapause eggs can break the diapause and restart the embryo development.The molecular mechanism and regulatory network of diapause and diapause termination of the Artemia Sinica have become a hot topic.NEDD4 is a HECT domain-containing E3 ubiquitin protein ligase,which can participate in the regulation mechanism of various cell life activities through K63 and K48-linked polyubiquitination,such as DNA repair protein transport,autophagy immunity and inflammation,and proteasome pathway.In addition,NEDD4 can also monoubiquitinated its substrates and catalyzed the little-known multi-ubiquitin of K6 and K27 linkages,indicating that NEDD4 plays a variety of regulatory roles through mono-ubiquitin and multi-ubiquitin.Therefore,this study conducted a series of studies on the role of As-NEDD4 in the reactivation of Artemia Sinica diapause embryos and the early development of embryos.In this study,a total of 2453 bp of the EST and 5’ UTR of nedd4 gene were obtained for the first time by homologous cloning and RACE technology,which contained the complete domain sequence with the maximum open reading frame of 2361 bp,coding 786 amino acids and the protein molecular weight of 90 KD.Bioinformatics analysis showed that As-NEDD4 was a hydrophilic non-secretory protein without signal peptide and transmembrane structure.RT-QPCR and Western Blot analysis showed that the transcription level and translation level expression pattern of NEDD4 were similar and showed paragraph-type expression characteristics during the diapause termination and early embryo development of Artemia Sinica.Such expression patterns were quite consistent with the embryo development at 0-5h,15-20 h and 40-72 h after diapause of Artemia Sinica.In addition,we also detected the expression of As-MAP1LC3,an important autophagy marker regulated by As-NEDD4,which showed the same expression pattern as As-NEDD4,and the interaction between the two was involved in the biogenesis of early embryonic development autophagosomes.However,the expression pattern of Akt,an important substrate of NEDD4,was not highly correlated with the expression of As-NEDD4.This may be due to the fact that As-NEDD4 mediated ubiquitination regulates the phosphorylation level of As-Akt,but does not regulate the total amount of As-Akt,so there was no significant correlation in the total amount.In the WB experiment under low temperature stress,the three proteins showed a trend of decreasing first and then increasing and then decreasing again with the decrease of temperature,which might indicate that these three important cellular proteins were involved in the response process of low temperature stress.The As-NEDD4 was subcellular mapped by immunofluorescence,the results showed that As-NEDD4 was expressed in all parts of the body and was co-located with the nucleus to a large extent.Based on relevant literature and our experimental results,it is reasonable to speculate that the autophagy,which has been reduced to a very low level in diapause eggs,may be regulated and activated by As-NEDD4 after the termination of diapause to participate in diapause termination and early embryonic development,but the specific regulatory mechanism needs to be further studied.