Expression Of Artemia Sinica As-NUPR1Protein In Development And High Salt Stress

Artemia sinica is a kind of small crutaceans living in high-salt waters, has a strong abilityto adapt to environmental extremes, and can resist extreme adverse conditions of high saltand low temperature that many creatures can’t adapt.As-NUPR1is a stress-related protein,which plays an important role in diapause during embryonic development in the brine shrimpArtemia sinica，and has a complex and diverse cellular functions. There is no report for theresearch of Artemia sinica As-NUPR1expression pattern in the different development periodand under the condition of salt stimulus.In this study, we obtained Artemia sinica As-nupr1198bp open reading frame sequencefragment using molecular cloning techniques. Analysis of bioinformatics suggested that itencodes66amino acids, molecular weight is7.9kDa, and the theoretical isoelectric point is10.34. The As-NUPR1protein contains a phospho-p8domain which is the symbol structure ofthe NUPR1protein. Phylogenetic tree analysis showed that the similarity between Artemiasinica As-NUPR1and invertebrates was higher than vertebrate NUPR1, and evolutionaryrelationship was consistent with actual.Successful expression of As-NUPR1from the cDNA sequence isolated from A. sinicawas demonstrated using a prokaryotic expression system. The predicted molecular weight ofthe recombinant protein is15kDa, and the isoelectric point is7.17. The recombinant proteinis a solube protein. Purify protein with Histidine affinity chromatography. As-NUPR1polyclonal antibodies were prepared by immunization of Balb/c mice with purifiedrecombinant As-NUPR1protein as an antigen, and immunological studies were carried out.Detect the As-NUPR1spatial expression patterns in different development periods ofembryo with immunohistochemistry. The experimental results showed that the expression ofAs-NUPR1is widely distributed at different developmental stages in A. sinica, and there wasno tissue or organ specificity.Expression of As-NUPR1during different developmental stages of the embryo and inresponse to salinity stress was analyzed in A. sinica using western blots，the result shows thatexpression of As-NUPR1decreased gradually during the diapause termination stage ofembryo development, after which there was a general increase in expression after breakingthe shell. In addition, As-NUPR1expression was highly upregulated under conditions of highsalinity. These results suggest that the As-NUPR1protein is a stress-related protein that plays a role in protecting embryos from high salt damage in different embryonic developmentalstages, especially during the diapause period.