Identification of the glycerol kinase gene and its role in diapause embryo restart and early embryo development of Artemia sinica

Abstract

Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization by transferring a phosphate from ATP to glycerol, yielding glycerol 3-phosphate, which is an important intermediate for both energy metabolism and glycerolipid production. Artemia sinica has an unusual diapause process under stress conditions of high salinity, low temperature and lack of food. In the process, diapause embryos of A. sinica (brine shrimp) accumulate high concentrations of glycerol as a cryoprotectant to prevent low temperature damage to embryos. Upon embryo restart, glycerol is converted into glucose and other carbohydrates. Therefore, GK plays an important role in the diapause embryo restart process. However, the role of GK in diapause termination of embryo development in A. sinica remains unknown. In the present study, a 2096 bp full-length cDNA of gk from A. sinica (As-gk) was obtained, encoding putative 551 amino acids, 60.6 kDa protein. As a crucial enzyme in glycerol uptake and metabolism, GK has been conserved structurally and functionally during evolution. The expression pattern of As-gk was investigated by quantitative real-time PCR and Western blotting. Expression locations of As-gk were analyzed using in situ hybridization. As-gk was widely distributed in the early embryo and several main parts of Artemia after differentiation. The expression of As-GK was also induced by stresses such as cold exposure and high salinity. This initial research into the expression pattern and stress response of GK in Artemia provides a sound basis for further understanding of the function and regulation of genes in early embryonic development in A. sinica and the stress response.

Keywords: Artemia sinica; As-gk; DEPC; DIG; Diapause termination; Digoxigenin; FBP; G3P; GK; GKD; ISH; LSD; NJ; ORF; PBS; PI; PTS; RT-PCR; Stress response; diethylpyrocarbonate; fructose-1,6-bisphosphate; glycerol 3-phosphate; glycerol kinase; glycerol kinase deficiency; glycerol kinase gene of Artemia sinica; in situ hybridization; isoelectric point; least square difference; neighbor-joining; open reading frame; phosphate-buffered saline; phosphotransferase system; real-time PCR.