The Expression And Function Of As-UCH-L5 In The Process Of Diapause Embryos Restart Of Artemia Sinica

Artemia sinica is a small crustacean with important economic and research value,which produces diapause eggs under harsh environmental conditions,has obvious stress resistance.Diapause embryos will restart when the growth environment is suitable.The mechanism of occurrence and reactivation of Artemia sinica diapause has not been clarified and needs to be further studied.The ubiquitin proteasome system(UPP/UPS)is a conserved protein degradation pathway in eukaryotes,which participates in a variety of cell life activities in vivo,such as cell cycle regulation,DNA damage repair,transcription regulation and so on.Ubiquitin proteasome system mainly relies on the dynamic process of interaction regulation between ubiquitinase and deubiquitinase.UCH-L5 belongs to the deubiquitinase UCHs family,has deubiquitination activity,and is the only protein in the UCHs family that is associated with the proteasome system.Therefore,this study explored the role and function of UCH-L5 in the restart of diapause embryo development of Artemia sinica.In the present article,As-uch-l5 was cloned by RACE technology,and the full-length was1011 bp,the maximum open reading frame was 969 bp,encoding 322 amino acids,the predicted relative molecular weight of protein was about 36 k Da,and the theoretical value of isoelectric point was 5.54.Bioinformatics analysis showed that As-UCH-L5 was a nonsecretory hydrophilic protein.Semi-quantitative PCR experiment was used to analyze the m RNA level in 7 periods of normal development of Artemia sinica,the results showed that the expression of As-uch-l5 decreased gradually.Western Blot was used to analyze the levels of As-UCH-L5 and related proteins in 7 stages of normal development of Artemia sinica.The results showed that the expression level of As-UCH-L5 firstly decreased in 0h-10 h,then increased at 15 h,and increased sharply at the 3d day of development.The expression trend of related proteins As-β-catenin and As-c-Myc basically decreased at 0h-10 h first,and gradually increased at 15h-20 h,at 40h-3d,it began to gradually decline.Subcellular localization of As-UCH-L5 was carried out by immunofluorescence technology,the results showed that As-UCH-L5 was colocalized with the nucleus to a large extent,and was almost expressed in the whole cells of Artemia sinica without specificity,at the 3d of development,As-UCH-L5 expression was more prominent in the chest and intestine of Artemia sinica.In order to have a clearer understanding of the function of As-UCH-L5 in diapause of Artemia sinica,which was treated with As-UCH-L5 inhibitor bAP15,Western Blot technology explored the expression pattern of As-UCH-L5.The concentration gradient experiment determined that the inhibitor b-AP15 had the best effect on As-UCH-L5 when it was 5μM.The key developmental period(10h,15h)of Artemia sinica was detected at this concentration,the expression of As-UCH-L5 and the signaling pathway related proteins As-β-catenin and As-c-Myc showed that the expression trend of these three proteins was completely consistent during the critical development period of Artemia sinica,and inhibit experimental protein expression was significantly reduced,which showed that As-UCH-L5 played a regulatory role in the Wnt-β-catenin signaling pathway in the reactivation of diapause embryo development of Artemia sinica,and As-UCH-L5 has a positive regulatory effect on the Wnt-β-catenin signaling pathway.In order to further verify the role of As-UCH-L5 in the early embryonic development of Artemia sinica,we tested the hatchability in the critical developmental period of Artemia sinica under the action of inhibitor b-AP15,and the results showed that the hatchability of Artemia sinica was significantly reduced at 10 h and 15 h after the addition of inhibitor compared with the control group.It was proved that As-UCH-L5 plays a key role in the early embryonic development of Artemia sinica.This study is conducive to improving the molecular regulation mechanism of Artemia sinica deactivation and early embryonic development,and provides a reference for the molecular mechanism of reactivation of embryonic development in Artemia sinica.