Expression Pattern of Glycogen Phosphorylase Gene During Development and in Response to

Temperature
Author :ZhaoNa
Tutor:HouLin
School :Liaoning Normal University
CLC :S917.4
TYPE :Masters thesis
Download the PDF Full Text:http://www.topresearch.org/showinfo-177-678072-0.html
Year:2013
Abstract:
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 from25C to20C and was subsequently upregulated in the range15C to5C.
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.