斑马鱼akt3基因的克隆及其表达图谱与过表达分析

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

采用RT-PCR和RACE相结合的方法,克隆得到了斑马鱼的akt3/pkbγ基因,其cDNA全长为2874 bp,编码479个氨基酸。斑马鱼Akt3具有Akt家族成员间保守的PH结构域、催化活性结构域和调节结构域以及两个保守的磷酸化位点Thr305和Ser472。与已发表的人、大鼠、小鼠的Akt3氨基酸序列比较,相似性分别为95.8%、94.7%和95.4%。对斑马鱼早期胚胎进行RT-PCR检测显示,akt3在0—4hpf(hours post fertilization)含量水平较高,6hpf到12hpf降低至较低水平,16hpf后表达量开始逐渐上升,60hpf至96hpf则稳定在较高水平。原位杂交结果表明:akt3在2hpf至96hpf的胚胎中整体都有表达,没有组织特异性。在成鱼中,除鳃部外,akt3在所检测的其他各组织器官中均有表达,在脑部和卵巢表达量较高;利用显微注射持续表达myr-akt3 mRNA研究其功能充分性时结果显示,过量表达斑马鱼akt3 mRNA能使斑马鱼胚胎发育滞后且伴随着尾部短粗、体节模糊、尾末端膨大甚至严重缩短等不同程度畸形。而在斑马鱼myr-akt3注射组发育至24hpf时观察(以排除akt3造成的发育延迟的影响),发现注射过akt3的斑马鱼胚胎的脑部厚度较对照组显著增大,表明akt3对斑马鱼胚胎脑部尺寸发育有影响。

In this study,an akt3/pkbγ cDNA in zebrafish,Danio rerio,were isolated and identified by reverse transcrip-tion polymerase chain reaction and rapid amplification of cDNA ends（RACE） methods.The full length cDNA of ze-brafish 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 phos-phorylation 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 devel-opment stages by RT-PCR displayed that akt3 highly found at 0—4 hpf（hours past fertilization） and fell below the de-tective 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 ex-pressed 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 im-portantly,the embryonic brain thickness increased at 24hpf which indicated that akt3 was essential for normal embry-onic brain development.