【作者】 陈文；

【导师】 聂刘旺；

【作者基本信息】 安徽师范大学 ， 细胞生物学， 2007， 硕士

【摘要】 在对非哺乳类脊椎动物的性别决定与分化机制的研究中,两栖类的研究明显落后于其他类群。目前的研究资料表明,遗传基因可能是大多数两栖类性别发育的主要因素。中华大蟾蜍(Bufo bufo gargarizans)分布广泛,是国内研究两栖类时常用的实验动物,其性别决定、性别分化相关基因迄今未见报道。Dmrt和Sox基因家族是脊椎动物中参与发育的两大基因家族。本文研究了中华大蟾蜍的Dmrt基因和Sox基因,研究结果如下:1. Dmrt基因家族是一个与性别发育相关的基因家族。该家族成员都含有一个具有DNA结合能力的高度保守的DM结构域,在性别决定和分化发育的调控中担负着重要的功能。本文采用简并PCR技术,对中华大蟾蜍Dmrt基因的DM域进行了扩增、克隆、SSCP筛选和序列分析,结果获得了4个Dmrt基因家族成员的7条DM序列,且无性别差异。BLAST分析发现与人类相应DMRT蛋白氨基酸序列的一致性分别为97%,97%,93%,95%,100%,97%,91%,根据中华大蟾蜍拉丁学名,将其分别命名为BgDmrt1,BgDmrt2,BgDmrt3a,BgDmrt3b,BgDmrt3c,BgDmrt5a,BgDmrt5b。研究结果显示,在中华大蟾蜍基因组中存在着Dmrt基因家族的多个成员。系统发生分析表明,该基因家族至少具有7个成员基因,在脊椎动物和非脊椎动物中都具有高度的进化保守性。2. Sox家族成员的典型特征是编码的蛋白质都具有一个保守的基序HMG-box,可以与DNA序列特异性结合,促进基因的转录,从而在性别决定及胚胎发育中起着重要的调控作用。其成员之一Sox9基因在不同分类地位的脊椎动物中具有进化保守性,是一个重要的转录调控因子,参与性别发育及软骨等多种组织和器官的发育过程。本文运用RT-PCR从中华大蟾蜍精巢中分离到了Sox9基因,总长1448 bp,编码框全长1443 bp(481个氨基酸),其中在N端103~181位共79个氨基酸为高度保守的HMG盒,C端373~481共109个氨基酸为比较保守的TA域。系统树分析进一步证实了本文获得的是Sox9基因。将中华大蟾蜍Sox9蛋白序列与七种动物Sox9的氨基酸序列相比较发现,它们的同源性高达80%以上(如与人的同源性为81%,与日本粗皮蛙的同源性为92%),显示Sox9基因在进化中比较保守。在中华大蟾蜍的精巢中检测到Sox9 mRNA的表达暗示该基因可能参与了该物种的性别发育过程。3.构建cDNA文库及从中筛选克隆基因是获得基因的一种可靠方法。本文运用SMART技术构建了中华大蟾蜍精巢全长cDNA文库。提取精巢总RNA,用Clontech公司SMARTTM cDNA文库构建试剂盒反转录合成第一链cDNA,LD-PCR扩增获得全长双链cDNA;经SfiⅠ酶切、层析柱分离后,500 bp以上的片段与λTriplEx2载体连接并包装,建成原始文库。经鉴定原始文库滴度为2.21×10~6 pfu/ml,重组率为91%;文库扩增后的滴度为2.94×10~9 pfu/ml,重组率为93.7%。插入片段大小分布于0.4~2.0 kb之间,平均长度约为1.0 kb,说明已构建文库质量较高,为进一步筛选、克隆精巢特异表达基因奠定了基础。从该文库中克隆到了泛素延伸蛋白基因,全长561 bp,包含完整的5’和3’非编码区,编码128个氨基酸,即泛素的76个氨基酸后融合了52个氨基酸的核糖体L40蛋白。更多还原

【Abstract】 In the field of the mechanism of sex determination and differentiation, it is evident that the studies on amphibians are lagged behind other non-mammalian vertebrates. According to research data at present, genetic factors may play an important role in sexual development in most amphibians. The widespread Chinese toad, Bufo bufo gargarizans, is a kind of common experimental organisms. To date, its genes related sex determination and differentiation have not been obtained. The Dmrt gene family and the Sox gene family participate in the development process of vertebrates. In this paper, we studied the Sox and Dmrt genes of Chinese toad, the results were as follows:1. The Dmrt gene family involved in sexual development encodes putative transcription factors including a DNA-binding homology motif, the DM domain. In present paper, seven distinct Dmrt genes were cloned and sequenced from Chinese toad using highly degenerate primers. A database search for the cloned sequences revealed the following percentage identity with the homologous Dmrt genes of the human: BgDmrt1=97%, BgDmrt2=97%, three isoforms of BgDmrt3 (BgDmrt3a=93%, BgDmrt3b=95%, BgDmrt3c=100%) and two isoforms of BgDmrt5 (BgDmrt5=97%, BgDmrt5=91%). Based on similarities of amino acid sequences in the DM domain, phylogenetic tree of vertebrate and invertebrate Dmrt genes was reconstructed. These Dmrt genes were grouped into seven distinct subfamilies.2. Sox proteins, all of which harbors a HMG box, form a large class of transcriptional regulators implicated in the control of a variety of developmental processes. One member of this family, Sox9, is a transcription factor that plays critical roles in developmental processes including sex determination and chondrogenesis. In this study, we isolated the Chinese toad Sox9 coding sequence in adult testis tissue by RT-PCR. The Chinese toad Sox9 encoded a 481-amino acid (aa) protein containing a HMG-box from aa 103 to 181 in the N-terminus region and a TA domain from aa 373 to 481 in the C-terminus region. The deduced amino acid sequence of toad Sox9 showed 92.0% similarity to that of Rana rugosa and 81.0% similarity to that of Homo sapiens. Phylogenetic analysis further supported that our isolated gene was a true homolog of Sox9. The detection of Sox9 mRNA in the testis suggested that this gene might be involved in the gonad development of Chinese toad.3. Constructing and screening cDNA library is a reliable method to obtain new genes. A full-length cDNA library from the testis of Chinese toad was constructed with the SMART (switching mechanism at 5’end of RNA transcript) technique. Total RNA was extracted from the testis and reversely transcripted into full-length cDNA using PowerscriptTM reverse transcriptase. First-strand cDNA was amplified by long-distance PCR. After Sfi I digestion and Chroma spin-400 fractionation, cDNAs (>500bp) were ligated toλTriplEx2 vector and were packaged with Gigapack? III Gold Packaging Extract. The optimal primary library contained 2.21×10~6 pfu/ml clones and the amplified library had a titer of 2.94×10~9 pfu/ml, in which over 90% clones were recombinant and the average sizes of inserted cDNAs was about 1.0 kb. These results showed that the testis cDNA library can be used for screening genes. A full-length gene with 5’and 3’untranslated regions was isolated from the cDNA library. Sequence analysis showed that this 561bp cDNA encoded a 128-aa ubiquitin/L40e extension protein.更多还原