Glycogen serves as a metabolic reserve and is involved in macromolecular synthesis. Glycogen phosphorylase (GPase) is a key enzyme involved in intracellular glycogen catabolism, catalyzing the first step in glycogen degradation. In the diapause, GPase catalyzes glycogen into the closely related molecule, sorbitol. In this study, the full-length cDNA of the GPase gene (2,790 bp) was isolated from Artemia sinica for the first time by rapid amplification of cDNA ends technology. The GPase gene encoded a protein of 853 amino acids belonging to the Glycosyltransferase GTB type superfamily. The expression pattern and location of GPase were investigated at various stages during the embryonic development of A. sinica using real-time PCR and in situ hybridization. High GPase expression was detected at the 0 and 5 h stages. Subsequently, expression declined and was maintained at a low level during the stages from 10 to 40 h following by a small increase at day 3. Expression was downregulated at temperatures ranging from 25 to 20 °C and was subsequently upregulated in the range 15-5 °C. In situ hybridization assays showed wide distribution of the GPase gene during different developmental stages. From the results of this study, we conclude that the GPase gene expression is stress-related and might play an important role in Artemia development and metabolism.
As-NUPR1, a stress-related protein, plays an important role in post-diapause during embryonic development in the brine shrimp Artemia sinica. In the present study, successful expression of As-NUPR1 from the cDNA sequence isolated from A. sinica was demonstrated using a prokaryotic expression system. The recombinant protein consisted of 132 amino acids with a molecular weight of 15 kDa, and a predicted isoelectric point of 7.17. As-NUPR1 polyclonal antibodies were prepared by immunization of Balb/c mice with purified recombinant As-NUPR1 protein as an antigen, and immunological studies were carried out. Expression of As-NUPR1 during different developmental stages of the embryo and in response to salinity stress was analyzed in A. sinica using Western blots. The experimental results showed that the expression of As-NUPR1 is widely distributed at different developmental stages in A. sinica, and there was no tissue or organ specificity. Expression of As-NUPR1 decreased gradually during the diapause termination stage of embryo development, after which there was a general increase in expression after breaking the shell. In addition, As-NUPR1 expression was highly upregulated under conditions of high salinity. These results suggest that the As-NUPR1 protein is a stress-related protein that plays a role in protecting embryos from high salt damage in different embryonic developmental stages, especially during the post-diapause period.
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