尼羅羅非魚nr2f2的鑒定、表達及其在生殖中的功能研究
發(fā)布時間：2022-02-24 14:31
nr2f2又名coup-tfII,是核受體家族的重要成員,其對哺乳動物早期性別分化、雄性Leidig細胞發(fā)育以及雌性育性的維持具有重要作用。目前,尚無nr2f2與魚類生殖發(fā)育相關(guān)的報道。在本研究中,我們從尼羅羅非魚基因組中分離了兩個nr2f2基因,命名為nr2f2a和nr2f2b。分析了nr2f2a和nr2f2b在尼羅羅非魚各組織中的表達和性腺中的細胞定位,并通過特異性抑制劑和基因敲除初步探究了它們在羅非魚生殖中的功能。主要結(jié)果如下:（1）尼羅羅非魚nr2f2s的分離及克隆�；谀崃_羅非魚基因組和性腺轉(zhuǎn)錄組,分離得到兩個nr2f2基因,命名為nr2f2a和nr2f2b。通過設(shè)計特異性引物,對nr2f2a和nr2f2b的ORF進行了克隆。nr2f2a的ORF全長1254bp,編碼417個氨基酸;nr2f2b的ORF全長1299bp,編碼432個氨基酸。氨基酸序列分析發(fā)現(xiàn),同其它脊椎動物的Nr2f2一樣,尼羅羅非魚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）脊椎動物nr2f2的系統(tǒng)進化及共線性分析。通過NCBI和Ensembl分離了脊...
【文章來源】：西南大學(xué)重慶市211工程院校教育部直屬院校
【文章頁數(shù)】：93 頁
【學(xué)位級別】：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
【參考文獻】：
期刊論文
[1]Fish germ cells[J]. XU HongYan1, LI MingYou1, GUI JianFang2 & HONG YunHan1,2 1Department of Biological Sciences, National University of Singapore, Singapore 119260, Singapore; 2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Science China（Life Sciences）. 2010(04)
[2]鯉魚精子超微結(jié)構(gòu)的研究[J]. 尤永隆,林丹軍. 動物學(xué)研究. 1996(04)
[3]長吻鮠精巢及精子結(jié)構(gòu)的研究[J]. 張耀光,羅泉笙,鐘明超. 水生生物學(xué)報. 1993(03)
[4]西埔灣黃鰭鯛精子發(fā)生和形成[J]. 洪萬樹,張其永,倪子綿. 水產(chǎn)學(xué)報. 1991(04)
[5]棉酚甲酸對金魚精子質(zhì)膜的影響[J]. 管汀鷺,趙南明. 生物物理學(xué)報. 1990(03)
本文編號：3642917
【文章來源】：西南大學(xué)重慶市211工程院校教育部直屬院校
【文章頁數(shù)】：93 頁
【學(xué)位級別】：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
【參考文獻】：
期刊論文
[1]Fish germ cells[J]. XU HongYan1, LI MingYou1, GUI JianFang2 & HONG YunHan1,2 1Department of Biological Sciences, National University of Singapore, Singapore 119260, Singapore; 2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Science China（Life Sciences）. 2010(04)
[2]鯉魚精子超微結(jié)構(gòu)的研究[J]. 尤永隆,林丹軍. 動物學(xué)研究. 1996(04)
[3]長吻鮠精巢及精子結(jié)構(gòu)的研究[J]. 張耀光,羅泉笙,鐘明超. 水生生物學(xué)報. 1993(03)
[4]西埔灣黃鰭鯛精子發(fā)生和形成[J]. 洪萬樹,張其永,倪子綿. 水產(chǎn)學(xué)報. 1991(04)
[5]棉酚甲酸對金魚精子質(zhì)膜的影響[J]. 管汀鷺,趙南明. 生物物理學(xué)報. 1990(03)
本文編號：3642917
本文鏈接：http://sikaile.net/shoufeilunwen/nykjbs/3642917.html
最近更新
教材專著
