nr2f2又名coup-tfII,是核受體家族的重要成員,其對(duì)哺乳動(dòng)物早期性別分化、雄性Leidig細(xì)胞發(fā)育以及雌性育性的維持具有重要作用。目前,尚無(wú)nr2f2與魚(yú)類(lèi)生殖發(fā)育相關(guān)的報(bào)道。在本研究中,我們從尼羅羅非魚(yú)基因組中分離了兩個(gè)nr2f2基因,命名為nr2f2a和nr2f2b。分析了nr2f2a和nr2f2b在尼羅羅非魚(yú)各組織中的表達(dá)和性腺中的細(xì)胞定位,并通過(guò)特異性抑制劑和基因敲除初步探究了它們?cè)诹_非魚(yú)生殖中的功能。主要結(jié)果如下:（1）尼羅羅非魚(yú)nr2f2s的分離及克隆�；谀崃_羅非魚(yú)基因組和性腺轉(zhuǎn)錄組,分離得到兩個(gè)nr2f2基因,命名為nr2f2a和nr2f2b。通過(guò)設(shè)計(jì)特異性引物,對(duì)nr2f2a和nr2f2b的ORF進(jìn)行了克隆。nr2f2a的ORF全長(zhǎng)1254bp,編碼417個(gè)氨基酸;nr2f2b的ORF全長(zhǎng)1299bp,編碼432個(gè)氨基酸。氨基酸序列分析發(fā)現(xiàn),同其它脊椎動(dòng)物的Nr2f2一樣,尼羅羅非魚(yú)Nr2f2a和Nr2f2b具有高度保守的DNA結(jié)合結(jié)構(gòu)域（DBD）和配體結(jié)合結(jié)構(gòu)域（LBD）。（2）脊椎動(dòng)物nr2f2的系統(tǒng)進(jìn)化及共線性分析。通過(guò)NCBI和Ensembl分離了脊... 

【文章來(lái)源】：西南大學(xué)重慶市211工程院校教育部直屬院校

【文章頁(yè)數(shù)】：93 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
ABSTRACT
中文摘要
Chapter 1 Literature review
    1 Overview of nuclear receptor superfamily
        1.1 Members of nuclear receptor superfamily and their unified nomenclature
        1.2 Structure of nuclear receptor
        1.3 Function of nuclear receptor
        1.4 Research progress of Nr2f2
    2 Gametogenesis in vertebrates
        2.1 Testis and spermatogenesis
        2.2 Ovary and oogenesis
    3 Purpose and significance of this study
Chapter 2 Isolation and expression of nr2f2 in Nile tilapia
    1 Introduction
    2 Materials and methods
        2.1 Experimental animal
        2.2 Main instruments and reagents
        2.3 Gene cloning
        2.4 Construction of phylogenetic tree
        2.5 Syntenic analysis
        2.6 RT-PCR and Real-time PCR
        2.7 Transcriptome analyses
        2.8 In situ hybridization
        2.9 Antibody preparation
        2.10 Western blot
        2.11 Immunofluorescence
    3 Results
        3.1 Cloning of nr2f2a and nr2f2b in Nile tilapia
        3.2 Amino acid sequence alignment of Nr2f2a and Nr2f2b
        3.3 Identification of nr2f2 in vertebrates
        3.4 Phylogenetic analysis of nr2f2 in vertebrates
        3.5 Syntenic analysis of nr2f2 in vertebrates
        3.6 Tissue distribution of nr2f2a and nr2f2b in Nile tilapia
        3.7 Ontogenic expression of nr2f2a and nr2f2b in Nile tilapia gonad
        3.8 Cellular localization of nr2f2a and nr2f2b by ISH
        3.9 Verification of Nr2f2a and Nr2f2b specific antibodies by Western blot
        3.10 Cellular localization of Nr2f2a by Immunofluorescence
    4 Discussion
        4.1 nr2f2a and nr2f2b originated from the fish specific whole genome duplication
        4.2 nr2f2b experienced a secondary loss in some basal group teleosts
        4.3 The expression of nr2f2a and nr2f2b in Nile tilapia
Chapter 3 Functional study of nr2f2s in Nile tilapia
    1 Introduction
    2 Materials and methods
        2.1 Experimental animal
        2.2 Main instruments and reagents
        2.3 Inhibitor administration
        2.4 Target site designing for mutation
        2.5 Synthesis of gRNA and Cas9 mRNA and microinjection
        2.6 Mutation detection and establishment of mutant line
        2.7 Egg and sperm quality assessment
        2.8 Embryonic development observation
    3 Results
        3.1 Inhibition of nr2f2 resulted in aberrant gametogenesis in Nile tilapia
        3.2 Disruption of nr2f2a and nr2f2b by CRISPR/Cas9
        3.3 Establishment of nr2f2a and nr2f2b mutant lines
        3.4 Haploinsufficiency of nr2f2 in female tilapia resulted in decreased egg quality
        3.5 Offspring of nr2f2 female heterozygotes displayed aberrant embryogenesis
    4 Discussion
        4.1 Both nr2f2a and nr2f2b are involved in Nile tilapia oogenesis
        4.2 nr2f2a is critical while nr2f2b is dispensable for Nile tilapia oogenesis
        4.3 nr2f2a is important for Nile tilapia testis development and spermatogenesis
Chapter 4 Conclusions
References
Acknowledgements
Publications during Ph.D.study
Research activities during Ph.D.study


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期刊論文
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