The Fuction And Expression Analysis Of EIF2?K1 In Embryo Diapause Termination Of Brine Shrimp,Artemia Sinicain

Artemia sinica is a small crustacean living widely in high saline water in China.Due to its faster development speed and unique embryo development pattern,it has become the main experimental material for genetics,embryology and molecular biology,and has important research value.A.sinica has a character of strong resistance to stress.When it encounters an unfavorable environment,its embryonic development will stagnate in the gastrulation stage,forming a special diapause embryo.When the environment is suitable,the diapause can be released and embryo restart developement.The e IF2? phosphorylation can response to various cellular stress.When cells meet with various external environmental stresses,protein synthesis can be regulated by phosphorylating the Ser51 site of the translation initiation factor e IF2?,resulting in reduced synthesis of e IF2-GTP-Met-t RNAi ternary complex and attenuated m RNA translation.eIF2?k1 can respond to various stress(including oxidative stress,protoheme deficiency,osmotic pressure,and heat shock)to inhibit protein synthesis at the initiation level of translation.Under the stress of different environmental factors on A.sinica and the restart of diapause embryos,As-eIF2?k1 plays an important role,but its molecular regulatory mechanism is still unclear.In this study,the full-length As-eIF2?k1 c DNA was successfully cloned using RACE technology for the first time,with a length of 1447 bp,including an open reading frame of969 bp,encoding 322 amino acids,298 bp 5'-UTR and 150 bp 3 '-UTR,protein molecular weight is 36.5 KDa.Bioinformatics analysis shows that As-eIF2?k1 is mainly located in the nucleus,and is a non-transmembrane hydrophilic protein.Immunofluorescence results showed that As-eIF2?k1 had no tissue or organ specificity in the expression site of A.sinica at various developmental stages.Quantitative real-time PCR analyzed the relative expression of As-EIF2?K1 from the gene level.During the development of A.sinica embryos,there was a gradual increase trend,and the expression level of this gene was the highest at 15 hours,and then it had a downward trend.Western blotting analyzed the expression levels of As-EIF2s1,As-ATF6,As-ATF4,and As-EIF2?K1 at different developmental stages of A.sinica at the protein level,and the results showed that the protein was expressed at the period of A.sinica early diapause and pseudimago.At the same time,the protein expression level under high salt and low temperature environmental stress is highly upregulated,which may be involved in regulating the endoplasmic reticulum stress response.In the RNA interference experiment,the A.sinica embryos in the experimental group appeared significant growth retardation.The experimental results show that eIF2?k1 plays an important role in the early embryonicdevelopment,diapause relief,protein translation regulation and environmental stress of A.sinica.This article analyzes the role of As-eIF2?k1 in the molecular regulation mechanism of A.sinica diapause embryo restart and resistance to adverse environmental factors.This study can enrich the theory of A.sinica embryo diapause and restart,and for other animals diapause restart research of molecular mechanism provides scientific basis,has important theoretical significance,and has potential application value for the development of A.sinica protein resources.