【作者】 宋娇莲；

【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】 脊椎动物线粒体DNA是共价闭合的双链分子,长度约为15~22kb。线粒体因其基因组结构简单,呈母系遗传等特点,已被广泛应用于分析动物个体和动物类群间的系统发生关系。龟鳖类是形态学上最特化的一支爬行动物,近年来,以线粒体基因组(mtDNA)为对象研究龟鳖类系统进化被研究者们广泛采用。目前,已测定20种龟鳖类动物的线粒体基因组全序列,这20种动物中,侧颈龟亚目仅1种,曲颈龟亚目19种,涉及到5个科:陆龟科10种,鳖科2种,淡水龟科4种,龟科、平胸龟科和海龟科各1种。除平胸龟和扁陆龟外,其余18种龟鳖类动物的mtDNA的基因排布方式都与典型的脊椎动物线粒体相同,非常保守。水龟组(Clemmys complex)由旧大陆潮龟科的拟水龟属、眼斑龟属和新大陆龟科的水龟属组成,这三个属以前一直被认为是同属的,但没有得到形态学和染色体等方面的认同。为了进一步研究,本文参照近源物种的线粒体基因组,设计了特异引物,采用PCR产物直接测序法测得了黄喉拟水龟(Mauremys mutica)和四眼斑水龟(Sacalia quadriocellata)线粒体基因组全序列。结果表明:黄喉拟水龟和四眼斑水龟线粒体基因组序列全长分别为16 609 bp和16 816 bp,均包括2个rRNA基因、22个tRNA基因、13个蛋白质编码基因和2个非编码区。为探讨水龟组三种属之间的关系,本文选用了进化较快的NADH4(NADH脱氢酶亚基)蛋白编码基因和相邻的tRNA基因(共985bp)构建系统进化树来探讨由淡水龟组成的水龟类的关系。结果显示,MP树支持黄喉拟水龟和四眼斑水龟构成姐妹群关系,同属于淡水龟科,并支持淡水龟科和陆龟科非姐妹群,都为单系起源。另外,基于12种龟类,2种鸟类,2种鳄类,2种有鳞类和一种哺乳动物的mtDNA的12个重链编码蛋白质的基因构建了MP树和ML树。比对的核苷酸有10 799个位点,包括6 237个简约信息位点,通过建立起来的系统进化树可以确认龟类与祖龙类(包括鸟类和鳄类)的亲缘关系比有鳞类近,推测龟类有可能起源于二叠纪和三叠纪时期具有双颞孔的古龙类祖先,随着进化,头盖骨逐渐消失。系统分析也表明,鳖科按照其传统学位置和其它隐颈龟组合形成曲颈龟亚目。更多还原

【Abstract】 In vertebrate animals, mitochondrial DNA (mtDNA) is generally a 15~22 kb circular genome containing 37 genes: 13 protein-coding genes, two rRNA genes, and 22 tRNA genes, as well as a control region. In recent years, mtDNA has been widely used as a useful marker system in numerous phylogenetic analyses of vertebrate relationships because of its maternal mode of inheritance and relative lack of recombination.Turtles have highly specialized morphological characteristics. To date, complete mtDNA genomes have been determined for only 20 species, including 19 cryptodiran turtles and a side-necked turtle.The current taxonomic status of the Clemmys complex of freshwater turtles places the Old World genera Mauremys and Sacalia in the Geoemydidae and the New World genus Clemmys in the Emydinae. These three genera were previously considered to be congeneric, but it was not supported by morphology and chromosome. To address this issue, we sequenced the complete mitochondrial DNA (mtDNA) for the Asian yellow pond turtle (Mauremys mutica) and four eyed-spotted turtle (Sacalia quadriocellata). DNA sequencing was based on the PCR fragments from primer pairs that were designed on the basis of mtDNA sequences of the related turtle species. The genome is 16 609 bp and 16 816 bp, respectively, containing 37 genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) and a putative control region, a situation that is similar to that found in other vertebrates. The gene organization of the mtDNA studied is typical for vertebrates such as placental mammals, other turtles and skinks, but distinct from that of alligators and snakes.We chose rapidly evolving ND4, the protein-coding gene, which was valuable in lower level questions. We examined the phylogenetic relationships of the Clemmys complex of the freshwater turtles using 985bp of the DNA encoding ND4, a mitochondrial gene and its adjacent tRNA genes. Our results indicated that Mauremys and Sacalia appeared to be sister taxa in the Bataguridae, and suggested the monophyly for the Bataguridae and the Emydidae based on the maximum parsimony (MP) phylogenetic trees. Our studies also suggested that the Bataguridae is not the sister-group with the Emydidae.Furthermore, we conducted maximum parsimony analysis and maximum likelihood using nucleotide sequences of 12 Heavy-strand proteins from twelve turtles, two birds, two crocodilians, two lepidosaurias and one mammal. Analysis of 10 799 aligned nucleotide positions (6 327 informative) led us to hypothesize that turtles are closely related to archosaurs (birds and crocodilians), supporting Tree (((birds, crocodilians), turtles), squamates). This is a significant molecular phylogeny on the placement of turtles relative to the archosaus and lepidosaus. It is likely that turtles originated from a Permian-Triassic archosauromorph ancestor with two pairs of temporal fenestrae behind the skull orbit that were subsequently diminished. Phylogenetic analyses also indicated that group of Trionychoidae was in their more traditional location as the sister taxon to all other hidden-necked turtles, collectively forming the Cryptodira.更多还原