Expression of Artemia Sinica As-NUPR1Protein in Development and High Salt Stress

Author :ZhangRui
Tutor:HouLin
School :Liaoning Normal University
CLC :S917.4
TYPE :Masters thesis
Download the PDF Full Text:http://www.topresearch.org/showinfo-177-678071-0.html
Year:2013
Abstract:
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 cant 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 ofAsNUPR1is 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.