广义拟水龟属线粒体基因组系统发生学及龟类动物线粒体ND5基因适应性进化研究

发布时间：2018-04-02 01:26

本文选题：淡水龟　切入点：线粒体DNA　出处：《安徽师范大学》2016年博士论文

【摘要】：淡水龟科广义拟水龟属(Mauremys sensu lato)物种广泛分布于欧亚大陆。由于特殊的分布模式,其物种间的系统发生学一直是研究的热点。前人关于该类群的分子学研究多数是采用部分线粒体基因或核基因,不同分子学标记所得的结论存在争议,且该属物种的系统地理学以及物种起源的研究也仍处于空白。线粒体基因组具有高突变率,低重组率以及严格母系遗传等特质,常被作为理想的分子标记,广泛应用于生物的起源以及系统发生学关系的研究中。由于相对较快的进化速率,线粒体DNA的进化一直被认为不受选择压力的调控。然而,随着细胞能量代谢领域的不断深入研究,已明确线粒体DNA编码的蛋白在氧化呼吸电子传递过程中具有重要的作用,线粒体DNA的变异受到了选择压的调控。研究表明,线粒体DNA的突变与多种疾病密切相关。龟类物种线粒体基因组在基因结构上相对保守,仅在部分物种中出现了控制区倍增和蛋白编码基因位移的现象。这种特异型的线粒体基因组进化动力学以及其受到的选择压调控机制仍有待进一步研究。本研究通过测定地中海拟水龟(M.leprosa)、里海拟水龟(M.caspica)、希腊拟水龟(M.rivulata)和黄喉拟水龟(M.mutica)的线粒体全序列,结合NCBI上已有的序列数据,基于最大似然法(Maximum likelihood,ML),贝叶斯法(Bayesian inference,BI)重建广义拟水龟属内物种系统发生树;使用化石记录标定,推算该属物种分歧时间;结合相关地理学事件,重建祖先起源地。结合陆龟科、鳖科和淡水龟科相关物种ND5基因序列,通过荧光定量PCR技术和生物信息学分析,探讨龟类动物特异型的线粒体基因进化模式及其相关的适应性机制。研究的主要结果和结论如下:1、拟水龟属系统发生分析结果表明,原按形态学比较划分的四个属,乌龟属(Chinemys)、花龟属(Ocadia)、拟水龟属(Mauremys)和安南龟属(Annamemys)之间有着密不可分的并系关系,建议将原四属物种合并入一个扩充的属,即广义拟水龟属(Mauremys sensu lato)。分歧时间和祖籍地重建结果显示东南亚可能是该属物种最早的起源地,后期的物种形成过程中遗传漂变作用起到了主导地位。日本拟水龟的分歧可能起因于日本岛屿化活动。多态微卫星分析结果表明,东亚和东南亚拟水龟种群间没有明显的分化,可能存在广泛的杂交事件发生。线粒体分析结果和微卫星结果的不一致性,支持东南亚的黄喉拟水龟可能是杂交起源。2、截至2016年3月,已完成测序并释放的龟鳖动物线粒体全序列共有115条,涉及龟鳖目11个科,61个种。大多数龟类动物的线粒体基因组在基因结构上相对保守,然而部分物种中出现了控制区倍增和蛋白编码基因位移的现象。研究通过比较具有典型脊椎动物线粒体基因组特征的乌龟线粒体DNA以及高度变异的平胸龟线粒体DNA,采用实时定量PCR技术进行相对定量,探讨ND5基因位置转移以及控制区倍增对龟类动物线粒体基因表达量的影响。结合陆龟科、鳖科和淡水龟科相关物种ND5基因序列,估算高度变异的线粒体DNA所受的选择压,并模拟ND5亚基的蛋白质三维结构,探讨基因碱基序列的变异对能量代谢的影响。结果表明,与保守龟类动物线粒体DNA相比,高度特异化的线粒体基因组具有关联能量代谢的适应性意义。其ND5基因的重排以及控制区的倍增可能产生了新的线粒体转录模式,即双控制区线粒体共有三个转录启动始点,分别起始转录三条不同长度的转录本,通过ND5基因原初转录产物长度的缩短,导致其表达量的上调。而高水平的非同义替代率,可能增加了ND5蛋白的跨膜面积,从而增强其为合成ATP供给能量的能力。综上所述,本研究阐明了淡水龟科拟水龟属物种的系统发生关系以及历史性起源、物种形成事件,评估东亚东南亚拟水龟的基因流水平,进一步探讨其种间杂交程度。并通过分析龟类动物线粒体蛋白编码基因ND5的进化,探讨部分物种特异型的线粒体基因进化模式及其相关的适应性机制。
[Abstract]:Generalized quasi Clemmys Bataguridae (Mauremys sensu lato) species widely distributed in Eurasia. The specific distribution pattern, the system of species occurrence has been a focus of research. The previous studies on the molecular groups is the most part of mitochondrial genes or nuclear genes with different molecular markers, the conclusion is controversial the phylogeography of the origin of species and genus species are still blank. The mitochondrial genome has a high mutation rate, low recombination rate and strict maternal genetic traits, is often regarded as the ideal molecular markers, has widely used in biological origin and study the relationship between system. Due to the relatively fast rate of evolution, mitochondrial DNA evolution has been considered not be regulated by the selective pressure. However, with the deepening of research in the field of cellular energy metabolism, mitochondrial DNA has been clear Encoding protein plays an important role in the oxidative respiratory electron transfer process by regulating mitochondrial DNA variation in selection pressure. The results show that the mitochondrial DNA mutation associated with many diseases. The turtle species mitochondrial genome is conserved in the gene structure, only in some species appeared in the control area of multiplication and protein encoding the gene displacement phenomenon. The mitochondrial genome of the evolutionary dynamics of this specific type and selection by pressure regulation mechanism still needs further research. In this study, through the determination of Mediterranean turtle (M.leprosa), where the sea turtle turtle (M.caspica), Greece (M.rivulata) and mauremysmutica (M.mutica) of the complete sequence of mitochondrial, combined with the sequence data already on the NCBI, based on the maximum likelihood method (Maximum, likelihood, ML), (Bayesian inference, BI Bayesian method) reconstruction of generalized quasi species Clemmys system The occurrence of trees; use of fossil record calibration, the time between species differences; combined with the geography event, reconstruction of ancestral origin. Combined with the tortoise branch, kinosternidae Bataguridae and related species ND5 gene sequence by fluorescence quantitative PCR and bioinformatics analysis on mitochondrial gene evolution model for turtle animal specific type and the adaptive mechanism. The main research results and conclusions are as follows: 1, to Clemmys system analysis results show that the original four genera according to the morphological comparison of the division, the tortoise (Chinemys), is a flower turtle (Ocadia), quasi Clemmys (Mauremys) and Annan (Annamemys) between the turtle a close relationship and, suggested that the four species of the genus into an extended genus, namely generalized quasi Clemmys (Mauremys sensu lato). The different time and the ancestral reconstruction results in Southeast Asia may be the earliest species The origin, the late species form of genetic drift effect plays a dominant position. Japanese turtle differences may arise from the Japanese islands. Microsatellite analysis results show that the quasi East and Southeast Asia, no obvious differentiation of turtle populations, hybridization events may exist widely. Mitochondrial and micro analysis results the satellite is not consistent and support in Southeast Asia may be Huang Houni turtle hybrid origin.2, as of March 2016, has completed the sequencing and sequence of animal mitochondrial release of the turtles had a total of 115, involving testudinata 11 families and 61 species. Most turtles animal mitochondrial genome is relatively conservative in genetic structure, but some the species appeared in the control area and double protein encoding gene displacement phenomenon. Comparing with genomic characteristics of typical vertebrate mitochondrial turtle mitochondrial DNA And the height variation of turtle mitochondrial DNA, relative quantification using real-time quantitative PCR technique, ND5 gene transfer and control of position area double expression of turtle animal mitochondrial gene. Combined with the tortoise branch, kinosternidae Bataguridae and related species ND5 gene sequence, the estimation of mitochondrial DNA was highly variable the selection pressure, and simulate the three-dimensional structure of protein ND5 subunit gene sequence, to explore the influence of variation of energy metabolism. The results showed that compared with the conservative turtle animal mitochondrial DNA, mitochondrial gene was highly specific adaptive significance of associated with energy metabolism. The multiplication of ND5 gene rearrangement and control region may produce a new model of mitochondrial transcription, including mitochondrial double control zones of the three transcription start point, respectively, to initiate transcription of three different lengths of ND5 gene transcripts, through the original Shorten the length of the transcripts, the expression level was raised. And the high level of non synonymous substitution rate may increase the transmembrane area of ND5 protein, thereby enhancing its ability to synthesize ATP supply of energy. In summary, this study elucidates Bataguridae quasi Clemmys relations and historical origin of species occurrence system the speciation events, evaluation of East and Southeast Asia to investigate the level of gene flow, further explore the interspecific hybridization degree. And through the analysis of turtle animal mitochondrial gene ND5 encoding protein evolution, to explore the species specific type of mitochondrial gene evolution model and its adaptability mechanism.

【学位授予单位】：安徽师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q953

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