發(fā)布時(shí)間：2018-05-02 11:02

  本文選題：鯉魚 + 瓦氏雅羅魚　； 參考：《上海海洋大學(xué)》2016年博士論文

【摘要】：鯉科魚類原產(chǎn)于歐亞大陸、北美洲以及非洲,目前幾乎在世界各地均有分布。由于諸多原因,使得鯉科魚類、在各大洲的豐富度嚴(yán)重失衡,亞洲幾乎包括了所有鯉科亞類群的種類,含有最為豐富的物種多樣性,而歐洲的種類卻最為匱乏。位于東亞的中國(guó)包含十分豐富鯉科魚類,是世界上鯉科魚類種類最多的國(guó)家之一。鯉科魚類是一類淡水魚且主要生活在淡水環(huán)境,但也有一些種類能夠在鹽堿水環(huán)境中生存和繁殖。在世界范圍內(nèi),鯉科魚類中的鯉魚和瓦氏雅羅魚作為重要的水產(chǎn)經(jīng)濟(jì)物種得到了廣泛的關(guān)注。除去其經(jīng)濟(jì)價(jià)值,鯉魚和瓦氏雅羅魚也有十分重要的生物學(xué)意義,為一些科學(xué)問(wèn)題的研究和解決提供了良好的生物材料,例如:鯉魚作為異源四倍體生物,為研究基因的復(fù)制、染色體及基因組的加倍提供了重要模型;瓦氏雅羅魚具有較強(qiáng)的耐鹽堿性,為研究生物群體在高鹽堿脅迫環(huán)境下的系統(tǒng)發(fā)育關(guān)系以及耐鹽堿機(jī)制提供了重要的材料。鑒于鯉魚和瓦氏雅羅魚的重要經(jīng)濟(jì)價(jià)值和研究意義,本文對(duì)鯉魚和瓦氏雅羅魚開展了一系列的研究工作,主要包括以下三方面:1.鯉魚系統(tǒng)發(fā)育基因組學(xué)研究鯉魚(Cyprinus carpio)起源于歐亞大陸,是世界上養(yǎng)殖范圍最廣的淡水養(yǎng)殖經(jīng)濟(jì)魚類之一。由于鯉魚的養(yǎng)殖和馴化歷史悠久,再加上長(zhǎng)期的地理隔離等原因,造成鯉魚在全球范圍內(nèi)存在諸多亞種和品系。雖然這些差異為鯉魚的遺傳選育工作提供了豐富的種質(zhì)資源,但是由于鯉魚的繁育過(guò)程經(jīng)常會(huì)出現(xiàn)多重雜交和等位基因的“超入侵”等現(xiàn)象,往往造成不同的亞種和品系之間的遺傳背景復(fù)雜且模糊,出現(xiàn)系統(tǒng)發(fā)育關(guān)系和親緣關(guān)系不確定等問(wèn)題。這些問(wèn)題亟需應(yīng)用更加綜合的遺傳工具,針對(duì)更多樣化、更廣泛的研究對(duì)象來(lái)解決。在本研究中,我們通過(guò)對(duì)9個(gè)品系的26尾鯉魚進(jìn)行高通量全基因組測(cè)序,然后應(yīng)用生物信息學(xué)的方法提取線粒體基因組片段,拼接組裝得到線粒體全基因組序列。緊接著對(duì)線粒體全基因組進(jìn)行單倍型分析和系統(tǒng)發(fā)育關(guān)系分析,對(duì)Cyt b基因和d-loop區(qū)進(jìn)行系統(tǒng)發(fā)育關(guān)系分析。上述分析為解決鯉魚復(fù)雜的系統(tǒng)發(fā)育關(guān)系、鯉魚的種質(zhì)資源保護(hù)、新品種的遺傳選育和開發(fā)利用提供重要的參考依據(jù)。本研究解決和闡明的爭(zhēng)議性問(wèn)題如下:1.1歐洲鯉魚品系和亞洲鯉魚品系之間親緣關(guān)系較遠(yuǎn),應(yīng)該分屬于不同的亞種;1.2同屬于江西地區(qū)的興國(guó)紅鯉和荷包紅鯉親緣關(guān)系相對(duì)較近,與甌江彩鯉親緣關(guān)系較遠(yuǎn);1.3泰國(guó)鯉、日本錦鯉和甌江彩鯉親緣關(guān)系較近,泰國(guó)鯉和日本錦鯉很有可能均起源于中國(guó)的甌江彩鯉,或者這三種鯉魚品系具有共同的祖先種群;1.4首次從線粒體全基因組的角度推測(cè)了松浦鏡鯉的選育過(guò)程。明確了在起源于歐洲地區(qū)的德國(guó)鏡鯉品系基礎(chǔ)上培養(yǎng)和選育得到的松浦鏡鯉品系與歐洲亞種之間區(qū)別顯著,反而與東亞亞種聚為一類,尤其與興國(guó)紅鯉聚類最為緊密。提示松浦鏡鯉在我國(guó)本土化培育過(guò)程中,為了較快的適應(yīng)本土環(huán)境,可能以興國(guó)紅鯉作為母本與德國(guó)鏡鯉進(jìn)行了雜交選育,雜交得到的后代為了獲得德國(guó)鏡鯉的優(yōu)良性狀又不斷的與德國(guó)鏡鯉進(jìn)行回交,最后選育得到性狀穩(wěn)定遺傳的松浦鏡鯉;1.5明確在鯉魚品系間的系統(tǒng)發(fā)育分析中,d-loop可以替代線粒體全基因組描述其系統(tǒng)發(fā)育關(guān)系。為鯉科魚類或其他物種的系統(tǒng)發(fā)育研究提供了數(shù)據(jù)支持和借鑒意義。在國(guó)內(nèi)水產(chǎn)領(lǐng)域,首次應(yīng)用生物信息學(xué)方法從基因組數(shù)據(jù)中調(diào)取線粒體全基因組數(shù)據(jù)進(jìn)行分析研究,為以后的基因組學(xué)分析另辟蹊徑。2.瓦氏雅羅魚不同群體間的系統(tǒng)發(fā)育基因組學(xué)分析及抗逆性的研究瓦氏雅羅魚是雅羅魚亞科,雅羅魚屬的一個(gè)物種,廣泛分布在黑龍江流域各水系,在我國(guó)內(nèi)蒙古的一些內(nèi)陸湖也有少許分布。盡管瓦氏雅羅魚大部分是在淡水環(huán)境下生存,但是對(duì)于鹽堿性的生存環(huán)境也有極強(qiáng)的耐受性,例如,在著名的鹽堿湖——達(dá)利諾爾湖泊中就有瓦氏雅羅魚的分布。此外,從生態(tài)學(xué)的角度來(lái)看,達(dá)利諾爾湖泊中的瓦氏雅羅魚為生態(tài)遷徙的很多候鳥提供了充足的食物,在生態(tài)學(xué)上也意義重大。瓦氏雅羅魚因?yàn)槠漭^高的耐鹽堿性,也成為極端環(huán)境下水產(chǎn)領(lǐng)域的一個(gè)重要經(jīng)濟(jì)物種。雖然瓦氏雅羅魚具有重要的經(jīng)濟(jì)意義和生態(tài)價(jià)值,但是目前對(duì)于其耐鹽堿性的機(jī)制還是所知甚少,關(guān)于其生理學(xué)和遺傳學(xué)方面的研究更少。因此造成不同生活環(huán)境的瓦氏雅羅魚群體間的遺傳資源、系統(tǒng)進(jìn)化關(guān)系以及適應(yīng)性差異方面的知識(shí)掌握有限。針對(duì)上述問(wèn)題,筆者開展研究并得到以下主要內(nèi)容和結(jié)論:2.1首先應(yīng)用第二代測(cè)序技術(shù)和生物信息學(xué)的手段,分別獲取了15尾淡水生存環(huán)境(黑龍江和松花江交匯處)和15尾鹽堿水生存環(huán)境(達(dá)里諾爾湖)下的瓦氏雅羅魚的線粒體基因組;2.2分析了線粒體基因組的基本信息;然后應(yīng)用線粒體基因組的遺傳信息來(lái)進(jìn)行了單倍型分析、系統(tǒng)發(fā)育關(guān)系分析、分化時(shí)間評(píng)估以及核苷酸多態(tài)性分析,用來(lái)檢測(cè)不同棲息環(huán)境下的瓦氏雅羅魚群體之間的遺傳多樣性,耐鹽堿性機(jī)制以及系統(tǒng)發(fā)育關(guān)系。2.3結(jié)果表明,瓦氏雅羅魚在淡水環(huán)境下受到脅迫較小,線粒體基因組變化較快,群體進(jìn)化較快,分化時(shí)間范圍廣;鹽堿水生存環(huán)境下受到的脅迫較大,線粒體基因組變化較小群體進(jìn)化較慢,分化時(shí)間范圍更狹窄。2.4本研究為水產(chǎn)生物抗逆性的研究提供了重要參考,為生物逆性環(huán)境下的群體進(jìn)化和分化關(guān)系提供了理論研究依據(jù),為瓦氏雅羅魚的種質(zhì)資源保護(hù)和遺傳育種提供了重要的參考。3.鯉魚frizzled基因家族的研究frizzled基因家族(fzd)在人體內(nèi)編碼10個(gè)同源蛋白,這些同源蛋白通常位于質(zhì)膜上。fzd基因與生物體內(nèi)的三個(gè)主要信號(hào)通路相關(guān)并且大多數(shù)fzd基因具有不同的生理作用,是脊椎動(dòng)物生長(zhǎng)發(fā)育過(guò)程特別是細(xì)胞增殖和胚胎發(fā)育過(guò)程中的主要決定因素之一,具有重要的研究意義和研究?jī)r(jià)值。在此,我們從鯉魚全基因組數(shù)據(jù)庫(kù)以及多個(gè)轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中對(duì)鯉魚的fzd基因進(jìn)行了掃描、比對(duì)和注釋,研究了鯉魚fzd基因家族與其他脊椎動(dòng)物fzd基因家族的關(guān)系并且分析了該基因家族的共線性。最后,通過(guò)rt-pcr技術(shù)分析了成年鯉魚七個(gè)組織(腦、心、脾、肝、腎、皮膚和血液)以及受精后不同發(fā)育階段(0小時(shí)、12小時(shí)、24小時(shí)、36小時(shí)、48小時(shí)、3天、5天和7天)的fzd基因的表達(dá)。本研究主要進(jìn)行了fzd基因家族系統(tǒng)發(fā)生、進(jìn)化和表達(dá)方面的分析,驗(yàn)證了全基因組序列組裝和注釋結(jié)果,對(duì)鯉魚第四輪全基因組復(fù)制后基因的命運(yùn)也進(jìn)行分析探討。我們的研究結(jié)果為更好地理解鯉魚fzd基因家族、基因組多輪復(fù)制后的基因的命運(yùn)、fzd基因在不同組織和生長(zhǎng)發(fā)育早期階段的基因表達(dá)等方面的研究提供了重要的參考依據(jù)和借鑒價(jià)值。本文主要研究?jī)?nèi)容和結(jié)論如下:3.1在鯉魚中首次確定出26個(gè)fzd基因,明確了該基因家族在鯉魚基因組中進(jìn)行了顯著的基因擴(kuò)張,推測(cè)鯉魚基因組中發(fā)現(xiàn)的fzd基因擴(kuò)張是第四輪全基因組復(fù)制以及片段化復(fù)制的結(jié)果。3.2明確了在鯉魚基因組中全部26個(gè)fzd基因分別與陸生脊椎動(dòng)物的fzd基因同源,并沒(méi)有魚類特有的fzd基因。3.3比較發(fā)現(xiàn),在其他脊椎動(dòng)物中fzd基因并非大量存在。推測(cè)fzd基因的功能較為保守,復(fù)制后的fzd基因功能冗余不發(fā)揮作用,因此冗余的fzd基因在物種進(jìn)化的過(guò)程中逐漸丟失。3.4由于鯉魚經(jīng)歷的第四輪全基因組復(fù)制時(shí)間較短,故大量的FZD復(fù)制基因在鯉魚基因組中得到保留,但鯉魚中FZD6卻迅速丟失。這表明某些基因在全基因組復(fù)制后發(fā)生了加倍,但是由于其功能的過(guò)度保守性或者該基因大量表達(dá)造成毒害效應(yīng)等原因,使得該基因會(huì)在短時(shí)間內(nèi)快速丟失。3.5根據(jù)鯉魚FZD基因表達(dá)譜,我們推測(cè)原始基因能夠在生物的生長(zhǎng)發(fā)育過(guò)程中發(fā)揮原來(lái)的功能并廣泛表達(dá),而復(fù)制得到的基因卻只能執(zhí)行部分功能且只在發(fā)育的特定階段特異性表達(dá)。
[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.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S917.4

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本文編號(hào)：1833602