Expression Pattern Of Glycogen Phosphorylase Gene During Development And In Response To Temperature Stress From Brine Shrimp, Artemia Sinica

Artemia sinica is distributed widely and belongs to a kind of small crustacean. It has twoforms to reproduce, ovoviviparously or oviparously. Under the external environment stress,the Artemia can produce the dormant, encysted embryos in the oviparous form. The embryosare in a state of diapause at the late gastrula stage. It has an important significance to Artemiato resist the unfavourable environment and is benefit to population fecundity. In animals,glycogen serves as a metabolic reserve and is involved in macromolecular synthesis.Glycogen phosphorylase (GPase) is a key enzyme involved intracellular glycogen catabolism,catalyzing the first step in glycogen degradation. In the diapaused insects, glycogen is relatedto temperature and GPase catalyzes glycogen into the closely related molecule, sorbitol. Thestudies about GPase are not many in crustaceans; furthermore, the studies about GPase in theembryonic development and in response to temperature stress from Artemia were notinvestigated. We have done depth studies about GPase of Artemia in diapause, diapausetermination, early development and temperature stress processes. Therefore, this study canprovide a theoretical significance and a potential application value for further research onmolecular mechanism of diapause in Artemia and exploitation of Artemia resources.In this study, the full-length cDNA of the GPase gene was isolated from Artemia sinicaby RACE technology, and the sequence analysis was conducted by bioinformatics technology.The location of GPase was investigated at various stages during the embryonic developmentof Artemia sinica using in-situ hybridization, and the expression pattern of GPase wasinvestigated by real-time PCR.The resulting cDNA (2,790bp) contained a2,562-bp open reading frame, a61-bp5’-untranslated regions (UTR) and a167-bp3’-UTR. It predicted that the GPase geneencoded a protein of853amino acids containing a conserved phosphorylase domain. Theputative GPase protein belonging to the Glycosyltransferase GTB type superfamily had acalculated molecular mass of98.19kD, with a pI of5.67. The results of multiple proteinsequence alignment analysis and phylogenetic tree revealed that the GPase gene wasconservative in the evolutionary process. The in-situ hybridization results showed widedistribution that positive GPase mRNA signals were detected at every stage.The real-time PCR results showed that GPase was expressed at every stage, but hadremarkable differences. High GPase expression was detected at the0h and5h stages. Subsequently, expression declined and maintained at a low level, then had a small increase atday3.In temperature stress experiment, the expression was downregulated at temperaturesranging from25°C to20°C and was subsequently upregulated in the range15°C to5°C. Fromthe results of this study, we conclude that the GPase gene might play an important role inArtemia development and is temperature stress-related gene involved in regulating the abilityof cold resistance.