斑马鱼ATOH8基因的克隆、表达及初步功能研究

【作者】 周晶耀；

【导师】 姚纪花；

【作者基本信息】 复旦大学 ， 遗传学， 2009， 硕士

【摘要】 目前已发现很多碱性螺旋-环-螺旋(basic helix-loop-helix,bHLH)转录因子,它们参与神经元发生、肌细胞生成等过程的调控。Atonal是果蝇中发现的一种前神经元bHLH基因,参与眼的发育。math6是近年来在哺乳类发现的Atonal同源基因。研究表明,在小鼠中,math6在肾小球足细胞、神经元和胶质细胞形成以及胰腺细胞的分化过程中起重要作用,但是该基因在胚胎发育过程中的作用尚未阐明。我们在斑马鱼中克隆了math6的同源基因ATOH8,并进行了生物信息学分析,同时利用整胚原位杂交技术检测了斑马鱼胚胎ATOH8的表达情况。在此基础上,我们进行了ATOH8的过表达和吗啉反义寡核苷酸抑制(morpholino knock down)试验,对其功能进行了初步分析。斑马鱼ATOH8基因全长873 bp,编码一个含266个氨基酸的蛋白。序列比对分析表明,斑马鱼的ATOH8编码的蛋白产物与牛,人,大鼠和猴子ATOH8分别具有52%、51%、50%和46%的序列同源性。原位杂交结果显示ATOH8在斑马鱼胚胎早期广泛表达,从15体节时期开始,则渐渐集中在视网膜、小脑、体节腹侧部等。斑马鱼ATOH8的过表达导致体节严重畸形,视网膜分层受到一定的影响,主要体现在外网状层的缺如;ATOH8-MO反义抑制后,胚胎严重畸形,表现在胚胎视网膜明显变小,眼间距缩短,分层受影响,体节缩短变宽或融合,肌肉松弛,尾部向腹侧弯曲,围心腔膨大等症状,并伴有发育迟滞现象。通过切片分析及标记基因的原位杂交,进一步印证了前述结果。TUNEL分析表明,ATOH8的下调引起视网膜部分细胞的凋亡。这些结果提示:ATOH8参与斑马鱼视网膜细胞和体节肌肉细胞的发育分化,在视网膜和体节发育的调控过程中,ATOH8起着不可或缺的重要作用。更多还原

【Abstract】 ATOH8, a bHLH transcription factor, is reported to have some effects on the nervous system, kidney and endocrine pancrease development in mice. However, there is no report to suggest functions of ATOH8 in zebrafish to date. We have cloned the zebrafish ATOH8 gene (GenBank Accession No.EU272033), which contains 873 nucleotides and encodes a putative protein of 266 amino acids. Sequence analysis shows that its protein shares 52%, 51%, 50% and 46% identidy with the corresponding amino acid sequence in cattle, human, rat and monkey, respectively. We studied its expression pattern and found that zebrafish ATOH8 was a developmentally regulated gene and widely expressed at the early development stage and located at specific places such as retina and somite later, which indicated us that ATOH8 might play important roles during early embryogenesis in zebrafish. Therefore, we further studied the function of ATOH8 gene in zebrafish embryonic development. Over expression of ATOH8 caused runtish trunk, twisted tail and development delay, with the outer plexiform layer absent in the retina (6day). On the other hand, MO knock-down of ATOH8 resulted in reduced eye size, twisted or shortened trunk, fused or(and) widened somites and incurvate tails. Furthermore, TUNEL assay revealed that knock-down of ATOH8 induced retina cell apoptosis in the MO-treated embryos during zebrafish retina and brain development. Our results showed that ATOH8 is required and may play important roles during development of retina and somite.更多还原