尼罗罗非鱼nr2f2的鉴定、表达及其在生殖中的功能研究
发布时间：2022-02-24 14:31
nr2f2又名coup-tfII,是核受体家族的重要成员,其对哺乳动物早期性别分化、雄性Leidig细胞发育以及雌性育性的维持具有重要作用。目前,尚无nr2f2与鱼类生殖发育相关的报道。在本研究中,我们从尼罗罗非鱼基因组中分离了两个nr2f2基因,命名为nr2f2a和nr2f2b。分析了nr2f2a和nr2f2b在尼罗罗非鱼各组织中的表达和性腺中的细胞定位,并通过特异性抑制剂和基因敲除初步探究了它们在罗非鱼生殖中的功能。主要结果如下:（1）尼罗罗非鱼nr2f2s的分离及克隆。基于尼罗罗非鱼基因组和性腺转录组,分离得到两个nr2f2基因,命名为nr2f2a和nr2f2b。通过设计特异性引物,对nr2f2a和nr2f2b的ORF进行了克隆。nr2f2a的ORF全长1254bp,编码417个氨基酸;nr2f2b的ORF全长1299bp,编码432个氨基酸。氨基酸序列分析发现,同其它脊椎动物的Nr2f2一样,尼罗罗非鱼Nr2f2a和Nr2f2b具有高度保守的DNA结合结构域（DBD）和配体结合结构域（LBD）。（2）脊椎动物nr2f2的系统进化及共线性分析。通过NCBI和Ensembl分离了脊...
【文章来源】：西南大学重庆市211工程院校教育部直属院校
【文章页数】：93 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[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]鲤鱼精子超微结构的研究[J]. 尤永隆,林丹军. 动物学研究. 1996(04)
[3]长吻鮠精巢及精子结构的研究[J]. 张耀光,罗泉笙,钟明超. 水生生物学报. 1993(03)
[4]西埔湾黄鳍鲷精子发生和形成[J]. 洪万树,张其永,倪子绵. 水产学报. 1991(04)
[5]棉酚甲酸对金鱼精子质膜的影响[J]. 管汀鹭,赵南明. 生物物理学报. 1990(03)
本文编号：3642917
【文章来源】：西南大学重庆市211工程院校教育部直属院校
【文章页数】：93 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[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]鲤鱼精子超微结构的研究[J]. 尤永隆,林丹军. 动物学研究. 1996(04)
[3]长吻鮠精巢及精子结构的研究[J]. 张耀光,罗泉笙,钟明超. 水生生物学报. 1993(03)
[4]西埔湾黄鳍鲷精子发生和形成[J]. 洪万树,张其永,倪子绵. 水产学报. 1991(04)
[5]棉酚甲酸对金鱼精子质膜的影响[J]. 管汀鹭,赵南明. 生物物理学报. 1990(03)
本文编号：3642917
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