基于线粒体基因组的两种鲤科鱼类系统发育基因组学分析及鲤鱼Frizzled基因家族的研究

发布时间：2018-05-02 11:02

本文选题：鲤鱼 + 瓦氏雅罗鱼　；参考：《上海海洋大学》2016年博士论文

【摘要】：鲤科鱼类原产于欧亚大陆、北美洲以及非洲,目前几乎在世界各地均有分布。由于诸多原因,使得鲤科鱼类、在各大洲的丰富度严重失衡,亚洲几乎包括了所有鲤科亚类群的种类,含有最为丰富的物种多样性,而欧洲的种类却最为匮乏。位于东亚的中国包含十分丰富鲤科鱼类,是世界上鲤科鱼类种类最多的国家之一。鲤科鱼类是一类淡水鱼且主要生活在淡水环境,但也有一些种类能够在盐碱水环境中生存和繁殖。在世界范围内,鲤科鱼类中的鲤鱼和瓦氏雅罗鱼作为重要的水产经济物种得到了广泛的关注。除去其经济价值,鲤鱼和瓦氏雅罗鱼也有十分重要的生物学意义,为一些科学问题的研究和解决提供了良好的生物材料,例如:鲤鱼作为异源四倍体生物,为研究基因的复制、染色体及基因组的加倍提供了重要模型;瓦氏雅罗鱼具有较强的耐盐碱性,为研究生物群体在高盐碱胁迫环境下的系统发育关系以及耐盐碱机制提供了重要的材料。鉴于鲤鱼和瓦氏雅罗鱼的重要经济价值和研究意义,本文对鲤鱼和瓦氏雅罗鱼开展了一系列的研究工作,主要包括以下三方面:1.鲤鱼系统发育基因组学研究鲤鱼(Cyprinus carpio)起源于欧亚大陆,是世界上养殖范围最广的淡水养殖经济鱼类之一。由于鲤鱼的养殖和驯化历史悠久,再加上长期的地理隔离等原因,造成鲤鱼在全球范围内存在诸多亚种和品系。虽然这些差异为鲤鱼的遗传选育工作提供了丰富的种质资源,但是由于鲤鱼的繁育过程经常会出现多重杂交和等位基因的“超入侵”等现象,往往造成不同的亚种和品系之间的遗传背景复杂且模糊,出现系统发育关系和亲缘关系不确定等问题。这些问题亟需应用更加综合的遗传工具,针对更多样化、更广泛的研究对象来解决。在本研究中,我们通过对9个品系的26尾鲤鱼进行高通量全基因组测序,然后应用生物信息学的方法提取线粒体基因组片段,拼接组装得到线粒体全基因组序列。紧接着对线粒体全基因组进行单倍型分析和系统发育关系分析,对Cyt b基因和d-loop区进行系统发育关系分析。上述分析为解决鲤鱼复杂的系统发育关系、鲤鱼的种质资源保护、新品种的遗传选育和开发利用提供重要的参考依据。本研究解决和阐明的争议性问题如下:1.1欧洲鲤鱼品系和亚洲鲤鱼品系之间亲缘关系较远,应该分属于不同的亚种;1.2同属于江西地区的兴国红鲤和荷包红鲤亲缘关系相对较近,与瓯江彩鲤亲缘关系较远;1.3泰国鲤、日本锦鲤和瓯江彩鲤亲缘关系较近,泰国鲤和日本锦鲤很有可能均起源于中国的瓯江彩鲤,或者这三种鲤鱼品系具有共同的祖先种群;1.4首次从线粒体全基因组的角度推测了松浦镜鲤的选育过程。明确了在起源于欧洲地区的德国镜鲤品系基础上培养和选育得到的松浦镜鲤品系与欧洲亚种之间区别显著,反而与东亚亚种聚为一类,尤其与兴国红鲤聚类最为紧密。提示松浦镜鲤在我国本土化培育过程中,为了较快的适应本土环境,可能以兴国红鲤作为母本与德国镜鲤进行了杂交选育,杂交得到的后代为了获得德国镜鲤的优良性状又不断的与德国镜鲤进行回交,最后选育得到性状稳定遗传的松浦镜鲤;1.5明确在鲤鱼品系间的系统发育分析中,d-loop可以替代线粒体全基因组描述其系统发育关系。为鲤科鱼类或其他物种的系统发育研究提供了数据支持和借鉴意义。在国内水产领域,首次应用生物信息学方法从基因组数据中调取线粒体全基因组数据进行分析研究,为以后的基因组学分析另辟蹊径。2.瓦氏雅罗鱼不同群体间的系统发育基因组学分析及抗逆性的研究瓦氏雅罗鱼是雅罗鱼亚科,雅罗鱼属的一个物种,广泛分布在黑龙江流域各水系,在我国内蒙古的一些内陆湖也有少许分布。尽管瓦氏雅罗鱼大部分是在淡水环境下生存,但是对于盐碱性的生存环境也有极强的耐受性,例如,在著名的盐碱湖——达利诺尔湖泊中就有瓦氏雅罗鱼的分布。此外,从生态学的角度来看,达利诺尔湖泊中的瓦氏雅罗鱼为生态迁徙的很多候鸟提供了充足的食物,在生态学上也意义重大。瓦氏雅罗鱼因为其较高的耐盐碱性,也成为极端环境下水产领域的一个重要经济物种。虽然瓦氏雅罗鱼具有重要的经济意义和生态价值,但是目前对于其耐盐碱性的机制还是所知甚少,关于其生理学和遗传学方面的研究更少。因此造成不同生活环境的瓦氏雅罗鱼群体间的遗传资源、系统进化关系以及适应性差异方面的知识掌握有限。针对上述问题,笔者开展研究并得到以下主要内容和结论:2.1首先应用第二代测序技术和生物信息学的手段,分别获取了15尾淡水生存环境(黑龙江和松花江交汇处)和15尾盐碱水生存环境(达里诺尔湖)下的瓦氏雅罗鱼的线粒体基因组;2.2分析了线粒体基因组的基本信息;然后应用线粒体基因组的遗传信息来进行了单倍型分析、系统发育关系分析、分化时间评估以及核苷酸多态性分析,用来检测不同栖息环境下的瓦氏雅罗鱼群体之间的遗传多样性,耐盐碱性机制以及系统发育关系。2.3结果表明,瓦氏雅罗鱼在淡水环境下受到胁迫较小,线粒体基因组变化较快,群体进化较快,分化时间范围广;盐碱水生存环境下受到的胁迫较大,线粒体基因组变化较小群体进化较慢,分化时间范围更狭窄。2.4本研究为水产生物抗逆性的研究提供了重要参考,为生物逆性环境下的群体进化和分化关系提供了理论研究依据,为瓦氏雅罗鱼的种质资源保护和遗传育种提供了重要的参考。3.鲤鱼frizzled基因家族的研究frizzled基因家族(fzd)在人体内编码10个同源蛋白,这些同源蛋白通常位于质膜上。fzd基因与生物体内的三个主要信号通路相关并且大多数fzd基因具有不同的生理作用,是脊椎动物生长发育过程特别是细胞增殖和胚胎发育过程中的主要决定因素之一,具有重要的研究意义和研究价值。在此,我们从鲤鱼全基因组数据库以及多个转录组数据库中对鲤鱼的fzd基因进行了扫描、比对和注释,研究了鲤鱼fzd基因家族与其他脊椎动物fzd基因家族的关系并且分析了该基因家族的共线性。最后,通过rt-pcr技术分析了成年鲤鱼七个组织(脑、心、脾、肝、肾、皮肤和血液)以及受精后不同发育阶段(0小时、12小时、24小时、36小时、48小时、3天、5天和7天)的fzd基因的表达。本研究主要进行了fzd基因家族系统发生、进化和表达方面的分析,验证了全基因组序列组装和注释结果,对鲤鱼第四轮全基因组复制后基因的命运也进行分析探讨。我们的研究结果为更好地理解鲤鱼fzd基因家族、基因组多轮复制后的基因的命运、fzd基因在不同组织和生长发育早期阶段的基因表达等方面的研究提供了重要的参考依据和借鉴价值。本文主要研究内容和结论如下:3.1在鲤鱼中首次确定出26个fzd基因,明确了该基因家族在鲤鱼基因组中进行了显著的基因扩张,推测鲤鱼基因组中发现的fzd基因扩张是第四轮全基因组复制以及片段化复制的结果。3.2明确了在鲤鱼基因组中全部26个fzd基因分别与陆生脊椎动物的fzd基因同源,并没有鱼类特有的fzd基因。3.3比较发现,在其他脊椎动物中fzd基因并非大量存在。推测fzd基因的功能较为保守,复制后的fzd基因功能冗余不发挥作用,因此冗余的fzd基因在物种进化的过程中逐渐丢失。3.4由于鲤鱼经历的第四轮全基因组复制时间较短,故大量的FZD复制基因在鲤鱼基因组中得到保留,但鲤鱼中FZD6却迅速丢失。这表明某些基因在全基因组复制后发生了加倍,但是由于其功能的过度保守性或者该基因大量表达造成毒害效应等原因,使得该基因会在短时间内快速丢失。3.5根据鲤鱼FZD基因表达谱,我们推测原始基因能够在生物的生长发育过程中发挥原来的功能并广泛表达,而复制得到的基因却只能执行部分功能且只在发育的特定阶段特异性表达。
[Abstract]:Cyprinid fishes are native to Eurasia, North America and Africa and are currently distributed almost all over the world. For many reasons, the richness of cyprinid fishes is seriously unbalanced in all continents. Asia includes almost all the species of Cyprinus, the most abundant species diversity, and the most scarce in Europe. In East Asia, China contains very rich carp fishes and is one of the most common species of cyprinidas in the world. Cyprinid is a kind of freshwater fish and is mainly living in freshwater environment, but some species can also survive and reproduce in salt and alkali water environment. Carp and wagffish in carp are important in the world. The aquatic species of aquatic products have been widely concerned. Except for their economic value, carp and Vallis have very important biological significance to provide good biomaterials for the research and solution of some scientific problems, such as the common Tetraploid of the carp as a allotetraploid, to study the duplication of genes, chromosomes and genomes. The important model is provided. The valsalis Salis has strong salt tolerance and provides important material for the study of phylogenetic relationship and salt tolerance mechanism in the environment of high salt stress. In view of the important economic value and research significance of carp and wagwl, a series of carp and wagwl carp are carried out in this article. The research work mainly includes the following three aspects: 1. Cyprinus phylogenetic genomics studies the Cyprinus carp (Cyprinus carpio) originated from Eurasia and is one of the most widely cultivated freshwater aquaculture fishes in the world. The carp in the world has a long history of culture and domestication, and the long period of geographical isolation, resulting in the global scope of carp. Although these differences provide rich germplasm resources for the genetic breeding of carp, the genetic background of different subspecies and lines is often complicated and blurred because of the frequent occurrence of multiple crosses and the "super invasion" of the alleles. These problems are urgently needed to apply more comprehensive genetic tools to solve more diverse, more extensive research objects. In this study, we sequenced the high flux genome of 26 carp from 9 strains of carp and then used bioinformatics to extract the mitochondrial gene. The whole genome sequence of mitochondria was assembled and assembled. After the analysis of the haplotype and phylogenetic relationship of the whole mitochondrial genome, the phylogenetic relationship between the Cyt B gene and the D-loop region was analyzed. The above analysis could solve the complex phylogenetic relationship of the carp, the protection of the germplasm resources of the carp and the genetic selection of the new varieties. The controversial issues solved and clarified in this study are as follows: 1.1 the relationship between the European carp lines and the Asian carp lines is relatively distant, and should be divided into different subspecies; 1.2 the relationship between the red carp and the red carp in Jiangxi is relatively close to the relationship with the Oujiang color carp. More distant; 1.3 Thailand carp, Japanese carp and Oujiang color carp were closely related, and Thailand carp and Japanese carp probably originated from the Oujiang color carp in China, or the three carp lines had common ancestor population. 1.4 the breeding process of pine Cyprinus carp was speculated for the first time from the angle of mitochondrial genome. It was clear that it originated in Europe. The difference between the Pinus Cyprinus carp line and the European subspecies on the basis of the culture and selection of the German mirror carp lines in the region is distinct, but it is closely related to the subspecies of East Asia, especially with the cluster of the Xingguo red carp, which suggests that in the native culture of China, the Cyprinus carpio may be used as the red carp for the faster adaptation to the local environment. The mother and German mirror carp were bred by cross breeding. In order to obtain the excellent characters of the German mirror carp, the offspring of the German mirror carp were constantly rebred with the German mirror carp. Finally, the pine Cyprinus carp with stable heredity were selected. 1.5 in the phylogenetic analysis of the carp lines, D-loop could replace the whole genome of the mitochondria to describe its lines. It provides data support and reference for phylogenetic studies of cyprinidas or other species. In the field of domestic fisheries, the first application of bioinformatics to genomic data from genomic data was used to analyze the whole genome of the mitochondrial genome, and a new approach to the later genomics analysis of.2. wagffish is different. The study of phylogenetic genomics and stress resistance among groups. The valsalis is a subfamily of the subfamily of the genus salve, a species of the genus salve, widely distributed in various water systems in the Heilongjiang basin, and in some inland lakes in Inner Mongolia, China. Although the large part of the waghl fish is in the freshwater environment, it is salinized. The living environment also has a strong tolerance, for example, in the famous salt lake, Dali Noel lake, the distribution of the valerfish. In addition, from an ecological point of view, the Valerie in Dali Noor Lake provides adequate food for many migratory migratory birds, and is significant in ecology. Fish, because of its high salt and alkaline resistance, has also become an important economic species in the field of aquatic products. Although it has important economic significance and ecological value, the mechanism of its salt tolerance is little known at present, and there are fewer studies on its physiology and heritage. The knowledge of genetic resources, phylogenetic relationships and adaptability differences among the environmental vwh population is limited. In view of the above problems, the author studies and obtains the following main contents and conclusions: 2.1 first, the 15 freshwater living environment (black dragon) was obtained by using the second generation sequencing technology and bioinformatics. The mitochondrial genome of the valsalis valsalis under the living environment of the river and the Songhua River and the 15 tailed saline and alkaline water (Dali Lake); 2.2 the basic information of the mitochondrial genome was analyzed. Then the haplotype analysis, the system analysis, the differentiation time assessment and the nucleotide polymorphisms were carried out by the genetic information of the mitochondrial genome. Sex analysis was used to detect the genetic diversity, salt tolerance and phylogenetic relationship between the different habitats of different habitats, salt tolerance and phylogenetic relationship.2.3 results showed that the valsalis rodfish was less stressed in the freshwater environment, the mitochondrial genome changed rapidly, the population was faster, the time range was wide, and the saline alkali water environment was in the environment. The low evolution of the mitochondrial genome is slower and the time range of differentiation is narrower.2.4. This study provides an important reference for the study of aquatic bioresistance. It provides a theoretical basis for the relationship between population evolution and differentiation under the reverse environment, and the protection and genetic breeding of the germplasm resources of the vadai. The species provides an important reference to the.3. carp frizzled gene family. The frizzled gene family (Fzd) encodes 10 homologous proteins in the human body. These homologous proteins are usually located on the plasmalemma and the.Fzd gene is related to the three main signaling pathways in the organism and most of the Fzd genes have different physiological functions, which are the growth and development of vertebrates. The breeding process, especially one of the main determinants of cell proliferation and embryo development, has important research significance and research value. Here, we have scanned the Fzd gene of carp from the whole carp genome database and multiple transcriptome databases, compared and annotated the common carp Fzd gene family and other vertebrae. The relationship between the Fzd gene family of animals and the analysis of the co linearity of the gene family. Finally, the expression of the Fzd gene in seven tissues of adult carp (brain, heart, spleen, liver, kidney, skin and blood) and the expression of Fzd gene in different developmental stages (0 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days and 7 days) after fertilization were analyzed by RT-PCR technique. The analysis of the phylogenetic, evolutionary and expression aspects of the Fzd gene family has been carried out to verify the whole genome sequence assembly and annotation results, and to analyze the fate of the gene after the fourth round full genome duplication of carp. Our results are to better understand the fate of the gene in the Fzd base family of carp and the multiple copies of the genome. The Fzd gene provides important reference and reference value in the study of gene expression in different tissues and early stages of growth and development. The main contents and conclusions of this paper are as follows: 3.1 the first 26 Fzd genes were identified in carp, and it is clear that the gene family has made significant gene expansion in the carp genome. The Fzd gene expansion found in the carp genome is the result of fourth round full genome replication and fragment replication..3.2 clearly shows that all 26 Fzd genes in the carp genome are homologous to the Fzd gene of terrestrial vertebrates, respectively, and no specific Fzd gene.3.3 is found in the fish, and the Fzd gene is not a large number of other vertebrates in other vertebrates. It is conjectured that the function of the Fzd gene is more conservative, and the duplicated Fzd gene function redundancy does not play a role, so the redundant Fzd gene gradually loses.3.4 in the process of species evolution, because the fourth round full genome duplication time experienced by the carp is shorter, so a large number of FZD replication genes are retained in the carp genome, but the FZD6 in carp is in the carp genome. It has been lost rapidly. This indicates that some genes have doubled after full genome replication, but because of the excessive conservativeness of their functions or the toxic effects of the gene, the gene will quickly lose.3.5 in a short time, according to the gene expression profiles of the Cyprinus carp FZD gene, and we speculate that the original gene could grow in the biological growth. In the course of development, the original functions are widely expressed, while the replicated genes can only perform partial functions and express only at specific stages of development.

【学位授予单位】：上海海洋大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

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