【作者】 张晓燕；

【导师】 王昌留；

【作者基本信息】 鲁东大学 ， 细胞生物学， 2015， 硕士

【摘要】 文昌鱼是脊索动物门头索动物亚门的代表动物，介于无脊椎动物和脊椎动物之间，有着独特的分类地位，在研究脊椎动物起源和进化方面具有十分重要的作用。文昌鱼的基因组没有经过大规模复制，结构相对简单，文昌鱼作为进化研究的模式生物越来越被人们所看重。dax1基因也称作nr0b1，是核受体超家族的一个重要成员。dax1基因最早是在人中发现并分离出来的，它的重复可引起剂量敏感性反转综合症，它的突变可引起先天性肾上腺发育不全。随后在瓣鳃类、鱼类、两栖类、爬行类、鸟类和其他哺乳类的一些物种中发现也有dax1基因的存在。自1774年首次文昌鱼发现以来，至今仍没有关于其dax1基因的研究报道。本论文选取青岛文昌鱼作为实验材料，从NCBI上下载与文昌鱼亲缘关系较近的物种dax1基因的序列，进行同源性比较，依据保守序列设计特异性引物。以文昌鱼RNA作为模板，反转录出一段cDNA序列，再利用RACE技术进行5’RACE和3’RACE，扩增出长度为1536bp的完整cDNA序列，它含有一个长度为876bp的开放阅读框，可以编码292个氨基酸，5’非编码区为216bp，3’非编码区为441bp。以文昌鱼基因组DNA作为模板，根据已知DNA序列片段利用染色体步移技术进行5’端步移和3’端步移，扩增了5’端和3’端侧翼部分序列，获得了长度为2644bp的DNA序列。选取长度为250bp左右的cDNA片段，用地高辛标记后作为探针，进行文昌鱼组织切片原位杂交，结果显示，dax1基因在文昌鱼的性腺和鳃中均有表达，在肌肉中几乎不表达，没有明显的雌雄差异。更多还原

【Abstract】 Branchiostoma is a representative of cephalochordate. It has a uniqueclassification status that lies between invertebrate and vertebrate. Branchiostoma playsan important role in evolutionary origins of vertebrate. The genome of Branchiostomadoes not undergo duplication. Therefore, it has caught great attention of biologists, andrecently seems to be a model to study evolution of vertebrate.Dax1is also called nr0b1，which is a member of the nuclear receptor superfamily.It was initially isolated from human. Abnormal duplication of human dax1causesmale-to-female dosage-sensitive sex reversal, while mutations in the gene areresponsible for X-linked adrenal hypoplasia congenita. Dax1also exists in theLamellibranchia, Pisces, Amphibia, Reptilia, Aves and Mammalia.Branchiostoma was firstly found by a zoologist in1774. However, dax1inBranchiostoma has not been reported. Branchiostoma belcheri tsingtauense was usedfor all experiments. Specific primers were designed from the conserved regions ofdax1sequences. The dax1cDNA was synthesized from total RNA using RACE. It is1536bp long, with a predicted ORF encoding a protein of292amino acids. The5’UTR and3’ UTR are216bp and441bp，respectively. The dax1DNA was amplifiedfrom genomic DNA using genome walking. DIG labeled cDNA probe was synthesizedby PCR. The probe is approximately250bp. In situ hybridization showed that dax1isexpressed in the gonad and gill, but not in the muscle. Dax1did not show an apparentsexually dimorphic pattern in our study.更多还原