MOLECULAR CLONING, EXPRESSION AND OVEREXPRESSION ANALYSIS OF AKT3 (PKB) IN ZEBRAFISH

In this study, an akt3/pkb cDNA in zebrafish, Danio rerio, were isolated and identified by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of zebrafish akt3 comprised 2874 base pairs (bp) with an open reading frame (ORF) of 1440 bp, encoding 479 amino acids. Analysis of the deduced amino acid sequences revealed that zebrafish akt3 contained all three domains and two phosphorylation sites (Thr305 and Ser472) conserved among akt family members, and showed high similarity with known sequence from human akt3 (95.8%), rat akt3 (94.7%) and mouse akt3 (95.4%). Analyzing embryos of different development stages by RT-PCR displayed that akt3 highly found at 0-4 hpf (hours past fertilization) and fell below the detective sensitivity at 6-12 hpf. After 16 hpf, its expression began to elevation and maintained at relative high level from 60 hpf to 96 hpf. Whole mount in situ hybridization analysis showed that zebrafish akt3 expressed all over the embryo and had no special expression pattern. In adult fish, RT-PCR analysis of tissue distribution demonstrated that akt3 expressed in all tested tissue sample except gill, and highly expressed in brain and vary. Additionally, we fundamentally addressed the role of akt3 in zebrafish embryonic development by microinjecting overactivated myr-akt3 in early stage embryo. We found that overexpressing akt3 led to growth retardation in embryos and causes tail deformities. More importantly, the embryonic brain thickness increased at 24hpf which indicated that akt3 was essential for normal embryonic brain development.