The Role And Expression Pattern Of As-akt/pkb Gene During Diapause Termination Of Artemia Sinica

In this study, RACE technology was used to isolate cDNA sequence of the akt homologof Artemia sinica (As-akt). In situ hybridization technology and Real-time PCR technologywere used to study As-akt expression site and the expression of As-akt and As-pdk1duringembryonic development, salinity and temperature stress, respectively. Artemia sinica live inhigh salinity water worldwide, because of it contains high unsaturated fatty acids and highprotein content, it regards as important aquaculture species weaning food. A.sinica possessestwo developmental pathways: ovoviviparous and oviparous. In oviparous development,encysted gastrulae (cysts) are produced as diapause embryos (diapaused cysts) to combat dry,cold, ice and other harsh environments and reduce the adverse environmental impact toembryos, which metabolic activity are greatly reduced and accopanied with metabolismslowing down. Therefore, diapause plays an important role in the resistance to environmentalstress and the protection of embryo survival. The protein serine/threonine kinase Akt also known as protein kinase B (PKB), is one of the most important signaling molecules in the cell.akt functions in various processes of embryonic development including proliferation, motility,growth, glucose homeostasis, survival and apoptosis. But there was little studies on theexpression pattern of akt during diapause termination, embryonic development, high salinitystress and low temperature stress, more studies need to be done.Molecular cloning and bioinformatics experimental results show that the full-lengthcDNA sequence of the akt homolog of Artemia sinica (As-akt) is1,613-bp. The full-lengthcDNA of As-akt contains a1,416-bp open reading frame (ORF) that encodes a472aminoacid polypeptide. ClustalX and MEGA4.0were used to analyze the evolutionary relationshipsduring different species akt amino acids. And several online services were used to analyzeAkt signal peptidase, transmembrane region, hydrophibility and hydrophobility,phosphorylation sites and others.The predicted molecular mass and isoelectric point of theputative protein are54.41kDa and7.97, respectively.In situ hybridization revealed that As-akt is expressed throughout the embryo at0h to20h and in the head, chest, abdomen and appendages at40h,3d and5d. Real-time PCRanalysis showed that As-akt and As-pdk1(3-phosphoinositide-dependent protein kinase-1)were highly expressed at0h,3d and5d of embryonic development. As-akt and As-pdk1transcripts were highly upregulated by high salinity and low temperature stress. All of these findings suggest that As-akt plays an important role in study on themolecular mechanism of diapause termination in Artemia sinica and have an applied value forArtemia sinica resources.