Identification Of The Glycerol Kinase Gene And Its Expression Pattern In Diapause Embryo Restart Of Artemia Sinica

Artemia sinica is a small crustacean distributed worldwide in hypersaline waters over awide temperature range. Artemia has a strong resistance to environmental stress, andundergoes an unusual diapause process under conditions of high salinity, low temperature,lack of food et al, in which development of the embryo is suspended at gastrula stage.Diapause may be terminated under suitable conditions and embryos resume development.Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization bytransferring a phosphate from ATP to glycerol, yielding glycerol3-phosphate withmagnesium as a cofactor. Glycerol3-phosphate is an important intermediate for both energymetabolism and glycerolipid production. In the process of diapause, embryos of A. sinicaaccumulate high concentrations of glycerol as a cryoprotectant to prevent low temperatureand desiccation damage to embryos. Upon embryo restart, glycerol is converted into glucoseand other carbohydrates. Therefore, GK plays an important role in the diapause embryo restartprocess. However, the role of GK in diapause and its termination in embryo development of A.sinica remains unknown.In the present study, the full-length cDNA of As-gk in A.sinica was obtained andanalyzed by bioinformatics and molecular evolution methods. The full-length of As-gk is2096bp, containing326bp5’ UTR,114bp3’ UTR and an open reading frame of1656bp,which encodes a putative protein of551amino acids. It contains a typical glycerol kinasedomain that is a marker of the FGGY superfamily. As a crucial enzyme in glycerol uptakeand metabolism, GK has been conserved structurally and functionally during evolution as itwas indicated by multiple alignments. Expression locations of As-gk were analyzed using insitu hybridization. As-gk was widely distributed in the early embryo and several main parts ofArtemia after differentiation which was consistent with study in fruit flies and mammals. Theexpression pattern of As-gk was investigated by quantitative Real-time PCR and Westernblotting. The results indicated that As-gk had a relatively high expression during earlydevelopment of Artemia (10h), suggesting glycerol kinase played a vital role as therate-limiting enzyme in glycerol uptake at diapause termination. As the consumption ofglycerol, the transcription of As-gk declined. Salinity and temperature challenge resultsdemonstrated the expression of As-gk was upregulated when the salinity increased ortemperature reduced. It was proved sensitive to temperature and salinity, being astress-associated protein which could protect embryos from cold exposure and high salinity. This initial research into the expression pattern and stress response of GK in Artemiaprovides a sound basis for further understanding of the function and regulation of genes inearly embryonic development in A. sinica and the stress response, having potential applicationvalue for developing and utilizing Artemia resource.