The Regulation Of The As-eIF2B In The Redevelopment Of Diapause Embryos

Artemia sinica is a type of crustacean widely distributed in inland salt lakes in China.When A.sinica encounters harsh environments,it will form diapause embryos to resist bad environments.When the environmental conditions are suitable,the diapause of dormant eggs is released,and the embryos are restarted under appropriate conditions and the regulation of a large number of genes.Eukaryotic initiation factor 2B(eIF2B)is not only an important protein involved in the initiation of eukaryotic translation,but also a guanylate exchange factor that can enhance protein activity.It is in eukaryotic translation initiation factor 2(eIF2)played an important role in the formation of an active ternary complex.In this process,eIF2B can convert the GDP of the inactive state bound to eIF2 into an active GTP,that is,the form of eIF2·GTP,so that eIF2 can regain its function and participate in the next series of cellular activities.In order to maintain the steady state of the cellular environment.However,so far,the regulatory role of eIF2B in the redevelopment of A.sinica diapause embryos has not been reported.In this study,the RACE technology was used to clone the full-length cDNA sequence of As-eIF2B of A.sinica for the first time,with a total length of 1247bp.Among them,the length of the open reading frame is 915bp,the length of the 5'untranslated region is 24bp and the length of the 3' untranslated region is 308bp,encoding a total of 304 amino acids and the protein molecular weight is 34KDa.The results of bioinformatics analysis showed that As-eIF2B,as a non-secreted hydrophilic protein with no transmembrane function,is mostly located in the cytoplasm.It is speculated that the theoretical isoelectric point of As-eIF2B is 8.84 and the phosphorylation site is 25.Through molecular evolution analysis,As-eIF2B is a highly conserved protein,consistent with its evolutionary position in crustaceans.Real-time qPCR technology was used to analyze the relative expression of As-eIF2B.The results showed that during the development of A.sinica embryos,As-eIF2B expression gradually increased with embryonic development and reached the highest level at 20 hours after development and began to downregulate at its next development stage;using western blot technology to analyze As-eIF2B,As-eIF2? and As-ATF4 protein expression patterns,the results showed that during the development of A.sinica,the expression level showed an upward trend,and reached the highest value at 20h.The relative expression level of As-eIF2B showed a significant downward trend under low temperature stress.Immunofluorescence analysis showed that As-eIF2B was not tissue and organ specific at 20h and 3d.This paper describes the role of As-eIF2B in the regulation of the eIF2 pathway during the embryonic development stage of Artemia sinica,which provides a theoretical basis for further research on the release of diapause in Artemia sinica,and further eIF2B in invertebrates The expression model provides new research ideas.