【作者】 尤翠平；

【导师】 刘少军；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2012， 博士

【摘要】 杂交和多倍化是基因组进化的重要驱动力,是物种多样化和产生新物种的重要途径。人工多倍体鱼的研究不仅在水产育种上具有重要的研究价值,在生物学基础理论研究方面也具有重要的意义,对不同倍性鱼的分子生物学研究可以帮助理解进化中物种发生多倍化或基因组复制的遗传机理以及物种在多倍化发生早期的分子遗传状况,同时由于杂交多倍体鱼含有不同的基因组,使其成为很好的研究线粒体母性遗传和核质互作的模型。线粒体是真核生物的能量工厂,具有自己的遗传系统—线粒体基因组(mitochondrial DNA, mtDNA),其正常功能的运行需要线粒体基因组和核基因组的协同作用。线粒体DNA具有小型性、自主性和多态性等特点。对线粒体DNA的研究除了能揭示线粒体DNA的结构、基因表达及其功能外,还在核质互作、分子进化规律、物种起源与分化等方面具有重要的意义。本文应用多种分子生物学技术,结合相关的生物信息学知识对不同倍性鲫鲂的线粒体基因组和四倍体鲫鲤及其亲本肝脏转录组中的线粒体相关核基因进行了较系统的探讨和研究,并对两种鲤科鱼类翘嘴鲌和黄尾鲴的线粒体DNA全序列进行了系统分析。本论文的主要研究内容如下：1.对天然雌核发育红鲫、三倍体鲫鲂和四倍体鲫鲂的线粒体基因组全序列进行了测序,研究充分利用各种生物信息学软件,系统详细的阐明了这些不同倍性杂交鱼线粒体DNA的分子结构、碱基组成、基因组成和排列等特点。天然雌核发育红鲫、三倍体鲫鲂和四倍体鲫鲂线粒体基因组序列全长均为16580bp,与母本红鲫的线粒体基因组序列长度一致。通过与亲本红鲫和团头鲂线粒体基因组全序列的比较,我们发现无论是在线粒体基因组的结构还是全序列的同源性比较上,三种鲫鲂不同倍性杂交鱼的线粒体全基因组均与母本红鲫更相似,研究表明尽管经历了杂交和多倍化,不同倍性鱼的线粒体DNA仍然遵循母性遗传规律。同时结果也说明在线粒体基因组水平不能体现出不同倍性杂交鱼与父本团头鲂具有亲缘关系,这一点提示我们在利用线粒体基因组对鱼类特别是杂交鱼类进行系统关系研究时应该注意其有效性。2.以四倍体鲫鲂为母本以团头鲂为父本进行了四倍体鲫鲂的回交实验,在检测的三尾回交子代中有两尾三倍体鲫鲂和一尾五倍体鲫鲂。采用一对引物我们克隆了回交子代及母本四倍体鲫鲂的线粒体ND2基因,通过全序列比对和同源性分析,我们发现线粒体基因组的母性遗传方式在鲫鲂回交中仍然具有很强的稳定性,同时分别在一尾三倍体鲫鲂和五倍体鲫鲂中各发现两个A→G碱基替换,与其他16种鲤科鱼类相应位点的比较中我们发现这是两个容易发生碱基变异的位点,并且变异的形式多为A和G之间的替换。该研究进一步证实了雌性四倍体鲫鲂可以产生两种倍性的卵子,同时说明在杂交鱼线粒体DNA中不会发生大规模的变异,但是受杂交和多倍化的影响可能会在容易发生变异的位点发生少量的碱基变异。3.异源四倍体鲫鲤在线粒体DNA的遗传上遵循严格的母性遗传规律。本研究在四倍体鲫鲤及其亲本红鲫和鲤鱼的肝脏转录组数据库中,筛选出了四倍体鲫鲤94个contigs对应41个基因、红鲫136个contigs对应46个基因、鲤鱼86个contigs对应37个基因与线粒体呼吸链的四个蛋白复合物相关。将红鲫和鲤鱼的线粒体呼吸链蛋白相关基因建立数据库,在数据库中搜索四倍体鲫鲤的线粒体呼吸链蛋白相关基因,寻找与四倍体鲫鲤每个基因最匹配的亲本基因。结果表明,在搜索的最佳匹配基因中即有红鲫的基因又有鲤鱼的基因,说明两个亲本的核基因组都参与了线粒体复合物蛋白的合成,并根据所搜索到的各亲本基因的数目,初步判定红鲫基因组和鲤鱼基因组线粒体呼吸链相关核基因在四倍体鲫鲤中的表达没有明显的偏好性。采用相同的方法分析其他16种线粒体相关的蛋白和酶基因得到了相似的结果。以上分析表明红鲫线粒体基因组、红鲫核基因组和鲤鱼核基因组三个基因组都参与了线粒体功能的运作。同时,我们发现在筛选的所有四倍体鲫鲤的线粒体相关基因中与亲本红鲫和鲤鱼的线粒体相关核基因以及三种鱼与斑马鱼的线粒体相关核基因大部分并不是一对一的关系,基因树和氨基酸比对分析揭示这些基因很多都是多拷贝基因。此外,对10个基因的核苷酸分歧率分析和8个基因编码区的氨基酸序列同源性分析表明线粒体呼吸链相关核基因在红鲫、鲤鱼和斑马鱼之间都具有较高的同源性,说明和能量代谢相关的基因在鱼类中比较保守,这些基因的保守性可能是四倍体鲫鲤各基因组之间可以正常协调并相互适应的一个重要原因,从而确保其正常的生长发育,并能够稳定地繁殖后代。对四倍体鲫鲤、红鲫和鲤鱼肝脏转录组中线粒体相关基因的分析对杂交鱼类的核质互作和杂交衰败等研究具有重要的参考价值。4.通过PCR扩增测序获得了翘嘴鲌和黄尾鲴的线粒体基因组全序列,序列全长分别为16622bp和16630bp,翘嘴鲌和黄尾鲴的线粒体基因组的大部分区段与GenBank中现有的序列具有较高的同源性。其结构组成也与其他脊椎动物较为一致,包括13个蛋白编码基因,2个rRNA基因,22个tRNA基因和2个非编码区(1个控制区和1个轻链复制起始区)。在数据分析中我们对翘嘴鲌和黄尾鲴线粒体全基因组的碱基组成、蛋白质密码子利用情况以及tRNA和rRNA的二级结构进行了详细分析,并通过与其他几种鲌亚科和鲴亚科鱼类线粒体控制区的比较识别了翘嘴鲌和黄尾鲴线粒体控制区的3个保守区域。同时,基于翘嘴鲌和黄尾鲴连同另外40种鲤科鱼类的线粒体全基因组以虹鳟鱼为外群建立了NJ系统树,系统树初步显示了翘嘴鲌和黄尾鲴在鲤科鱼类中的分类地位。本文得到的翘嘴鲌和黄尾鲴的线粒体全基因组序列均是两种鱼类在GenBank中的第一条序列数据,为鲤科鱼类系统分类学研究增添了新的数据,为以两种鱼类为亲本的杂交子代的线粒体基因组研究及其线粒体相关基因的研究奠定了基础。更多还原

【Abstract】 Mitochondria are double-layered membrane-bound organelles, which are found in nearly all eukaryotic cells. They act as cellular energy suppliers and carry their own genetic material, the mitochondrial genome (mtDNA). As a unique cytoplasmic genetic system, mitochondrial DNA (mtDNA) is a small, self-replicating and diverse genome. The study on mitochondrial genome is significant in many biological issues, such as gene structure and function, gene expression regulation on time and space, nucleo-cytoplasmic interaction, the dynamics of molecular evolution, the origin of mitochondria, the origin and evolution of species, and so on.Artificial polyploidy fish induced by hybridization has been used in aquaculture to produce sterility or improve production. Meanwhile, they are also useful for investigating mechanism of ploidy variation, sex determination, and mode of inheritance of mitochondrial genome as well. Previously, we obtained the allotetraploid crucian carp by hybridization of red crucian carp and common carp, and obtained diploid natural gynogenetic red crucian carps (2nGRCC), triploid hybrids (3nRB) and tetraploid hybrids (4nRB) by hybridization of red crucian carp (RCC) and blunt snout bream (BSB). The point of this work was to analyze the genetic information of the mtDNA sequences of2nGRCC,3nRB and4nRB, and to analyze the expression information of the mitochondria-related genes in transcriptome of the allotetraploid crucian carp liver. Meanwhile, we also obtained and analyzed the complete mitochondrial genomes of two wild fish, Culteral alburnus and Xdnocypris davidi. The major results in this paper were presented as follows:1. We firstly reported the genetic information of the complete mitochondrial genomes in2nGRCC,3nRB and4nRB and compared them with the mitochondrial genomes of RCC and BSB which represented the parental fish. The complete mitochondrial DNA (mtDNA) sequences of these three polyploid hybrids were all16580bp in length, as same as their female origin (RCC). Analysis of mtDNA structures and sequence comparison indicated that the complete mitochondrial genomes of2nGRCC,3nRB and4nRB were all quite similar to that of RCC rather than BSB. The similarity and divergence analysis also showed that the genetic relationship between these hybrids and RCC were closer than that between them and BSB. These results indicated that the mitochondrial genomes of2nGRCC,3nRB and4nRB were still strictly maternal inherited, although they experienced hybridization and polyploidization. Meanwhile, we found that it could not determine the relative relationship between these hybrids and BSB at the mtDNA sequences level, which implied that it should be cautious when using mtDNA as molecular marker in phylogenetic studies.2. The backcross of female tetraploid hybrids of red crucian carp (♀) xblunt snout bream ((?)) and male blunt snout bream generated two kinds of polyploidy hybrids:two triploid hybrids and one pentaploid hybrid. We sequenced the mitochondrial ND2genes of the maternal4nRB and three hybrid offspring including two triploid hybrids (3nRB1and3nRB2) and one pentaploid hybrid (5nRB). The ND2genes of4nRBs,3nRB1,3nRB2and5nRB were all1047bp in length, encoding348amino acids. The similarity and divergence analysis indicated that the mitochondrial ND2genes of backcross hybrid offspring still stringently subjected to the maternal inheritance rule. Additionally, we observed two substitution of A→G in the mitochondrial ND2genes of3nRB1and5nRB, respectively. By comparison, we also found A<>G substitutions in the same sites in other16fish. The results indicated that the mtDNA genes of hybrid offspring couldn’t capture mutations at large degree, but they probably captured few mutations at hot mutation sites.3. The transcriptome of the allotetraploid crucian carp, red crucian carp and common carp liver obtained in the previous study gave us an opportunity to explore the coordination between the nuclear genome and the mitochondrial genome in hybrid offspring. In this work, we filtered94contigs including41respiratory-related genes of the allotetraploid crucian carp,136contigs including46respiratory-related genes of of red crucian carp and84contigs including37respiratory-related genes of common carp in their transcriptomes. By building respiratory-related gene database of red crucian carp and common carp transcriptomes and then performing balstn research of the allotetraploid crucian carp respiratory-related genes in the database, we found out39best matches in red crucian carp respiratory-related genes for allotetraploid crucian carp,40best matches in common carp respiratory-related genes. The results indicated that both nuclear genomes of red crucian carp and common carp involved the OXPHOS process of allotetraploid crucian carp. Beside the mitochondrial respiratory-related genes, we also detected some other mitochondria-related genes including some genes encoding proteins located in membrane of mitochondria and some genes encoding enzyme of mitochondria. The analysis of these genes showed the similar results to the previous results of respiratory-related genes.4. Culter alburnus and Xdnocypris davidi Bleeker are both popular fish in aquiculture. This study first reported complete mitochondrial genomes of these two fish. The entire mtDNA sequences of Culter alburnus and Xdnocypris davidi Bleeker were16622and16630bp in length, respectively. Similar to other cyprinidae fish, the complete mitochondrial genomes encoded the37genes consisting of13protein-coding genes, two rRNA genes,22tRNA genes and a non-coding control region. The characters of the molecular structure and gene organization of the mtDNA genomes in these two fish were described in detail including three conserved domains of D-loop regions. On the other hand, the NJ phylogenetic tree based on the complete mitochondrial genome of Culter alburnus, Xdnocypris davidi Bleeker and other40cyprinidae fish with oncorhynchus mykiss as outgroup were built, which showed that Culter alburnus and Xdnocypris davidi Bleeker had very close relationship. This work will provide genetic information for the breeding of these two fish, and will help to understand the further phylogenetic relationship of cultrinae and Xenocyprinae fish.更多还原