瘦素-黑皮素信号通路相关基因的变异及其对绵羊体尺和脂肪厚度性状的影响
发布时间：2023-02-21 13:55
绵羊是重要的畜种,它能够为我们提供羊肉、羊奶、羊皮和羊毛等多种产品。对于肉用绵羊,生长和繁殖是选育的主要目标。但是,与绵羊生长和产肉相关的遗传因素还没有完全确认。因此,研究调节控制生长和产肉性状的代谢通路和基因将为提高羊肉产量和质量提供新策略。瘦素-黑皮质素信号通路上的基因在体重调节和能量稳态中至关重要。该通路上的相关基因,尤其是黑皮质素4受体(MC4R)、信号转导和转录激活因子3(STAT3)和瘦蛋白(LEP)基因能够通过影响下丘脑的活动来调控摄食和能量消耗。基于在人类和其它物种中的相关报道的启发,本研究认为很有必要确定瘦素-黑皮质素信号通路相关基因上的变异和这些变异对绵羊体尺和体脂性状的影响,特别是阐明这些基因的结构和功能以及它们在绵羊不同组织中的表达量、鉴定那些位于MC4R基因启动子区上对该基因的转录调控起到重要影响的变异。在本研究中,我们选择了5个中国地方绵羊品种,总计551只绵羊作为样本。使用聚合酶链反应(PCR)直接测序方法检测了这些基因的变异。用一般线性模型(GLM)研究这些变异与各种体尺和体脂性状间的关联。我们收集了42只绵羊从出生到六月龄的七个组织(下丘脑,肾,肝,心...
【文章页数】：210 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
ABBREVATIONS
CHAPTER1 Introduction and Literature Review
1.1 Introduction
1.1.1 Problem Statement
1.1.2 Justification
1.2 Candidate Gene Approach and Genetic Association
1.3 Genetic Markers
1.3.1 Restriction Fragment Length Polymorphisms(RFLPs)
1.3.2 Microsatellites
1.3.3 Single Nucleotide Polymorphisms
1.4 SNP Discovery Methods
1.5 Use of SNP Markers in Industry
1.6 The Sheep Growth and Development
1.7 Fat Deposition and Meat Value
1.8 The Melanocortin System Genes
1.9 Central Neural Circuits Energy Homeostasis
1.10 A Leptin/Melanocortin Pathway
1.11 MC4R Gene Polymorphism in Livestock
1.12 STAT3 Gene Polymorphism in Livestock
1.13 Overview of Experiments,Hypotheses and Objectives
1.13.1 General Overview
1.13.2 Study One:The Leptin-Melanocortin System Modulates Rapid Growth And Muscular High-Yield Carcass In Sheep Through Adipocytokine Signaling Pathway
1.13.3 Study Two:Genetic Variations in the MC4R Gene Linked To Elevated Body Weight and Fat-Related Traits in Sheep
1.13.4 Study Three:STAT3 Gene Expression and Its Role in Sheep Body Weight and Fatness Modulation
1.13.5 Study Four:Single Nucleotide Polymorphisms in the Leptin Gene and Their Associations with Growth and Fat-deposition Traits in the Sheep
CHAPTER2 The Leptin-Melanocortin System Modulates Rapid Growth and Muscular High-Yield Carcass in Sheep Through Adipocytokine Signaling Pathway
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1 Experimental Animals
2.3.2 Function and Pathway Enrichment Analysis
2.3.3 Tissue Collection and Transcriptome Data Analysis
2.3.4 Single Nucleotide Polymorphisms(SNPs)Detection from Re-Sequenced Data
2.3.5 Analyses of Positive Selection
2.4 Results
2.4.1 GO Function and KEGG Pathway Enrichment Analysis
2.4.2 Polymorphism Analysis
2.4.3 Positive Selection of the Leptin-Melanocortin Genes by FDIST Analysis
2.4.4 Analysis of Positive Selection
2.4.5 Haplotype Structure
2.4.6 The Relative Transcript Abundance of the Genes Involved In the Leptin-Melanocortin Signaling Pathway
2.5 Discussions
2.6 Conclusion
CHAPTER3 Genetic Variations in the MC4R Gene Linked To Elevated Body Weight and Fat-Related Traits in Sheep
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Ethical Statement
3.3.2 Experimental Animals and Measurements
3.3.3 Blood Collection
3.3.4 Isolation of Genomic DNA
3.3.5 Evaluation of the quality,purity,and concentration of DNA
3.3.6 Designing of Primers
3.3.7 PCR Reaction Mixture
3.3.8 PCR Program
3.3.9 Checking of Amplified PCR Products
3.3.10 Identification of Single Nucleotide Polymorphisms(SNPs)
3.3.11 RNA Extraction and Real-Time q PCR
3.3.12 Sequence Characterization of the Potential Promoter Region of MC4R in Sheep
3.3.13 Promoter Cloning and Generation of Luciferase Reporter Constructs
3.3.14 Purification of PCR product by PCR Clean-Up System(Omega Bio-Tek,Inc.,Norcross,GA,USA)
3.3.15 Vector Ligation of Amplified Products
3.3.16 Competent Cell Preparation and Transformation
3.3.17 Confirmation of Positive Clones
3.3.18 Colony PCR
3.3.19 Plasmid Isolation from Positive Clones
3.3.20 Plasmid Digestion by Restriction Endonuclease Enzymes
3.3.21 Cell Culture,Transfection,and Luciferase Assays
3.3.22 Statistical Analysis
3.3.23 Association Analysis
3.3.24 Expression Analysis
3.4 Results
3.4.1 Single Nucleotide Polymorphism(SNP)Identification
3.4.2 SNP Variation in Potential Cis-Regulatory Elements of the MC4R Gene
3.4.3 Association Analyses
3.4.4 Linkage disequilibrium and Haplotype Analysis of the MC4R Gene
3.4.5 Sequence Analysis of the Promoter Region of MC4R Gene
3.4.6 Differential Expression of the MC4R Gene across Age and Sex
3.4.7 The Proximal Minimal Promoter Region of the MC4R Gene
3.5 Discussion
3.6 Conclusions
CHAPTER4 STAT3 Gene Expression and Its Role in Sheep Body Weight and Fatness Modulation
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.3.1 Sample Collection
4.3.2 Lipid Extraction-Soxhlet Method
4.3.3 PCR Amplification,Sequencing,and Genotyping
4.3.4 Sequence Analysis
4.3.5 Collection of RNA Samples
4.3.6 RNA Purification and c DNA Synthesis
4.3.7 RT-PCR Analysis of Expression Patterns
4.3.8 Immunohistochemical Localization and Analysis of STAT3
4.3.9 Statistical analysis
4.4 Results
4.4.1 The sequence of the sheep STAT3 gene
4.4.2 STAT3 Relative m RNA Expression in Sheep
4.4.3 Immunohistochemical Analysis
4.4.4 SNP Identification and Analysis of Its Association with Body Size Traits
4.4.5 Effect of the Polymorphisms on STAT3 Locus on Body Measurement and Fat Deposition Traits
4.4.6 Linkage Disequilibrium and Haplotype Analysis
4.5 Discussion
4.6 Conclusions
CHAPTER5 Single Nucleotide Polymorphisms in the LEP Gene and Their Associations with Growth and Fat-deposition Traits in the Sheep
5.1 Abstract
5.2 Introductions
5.3 Experimental Section
5.3.1 Materials and Phenotypic Data Collection
5.3.2 PCR Amplification and SNP Identification
5.3.3 SNP Identification and Genotyping
5.3.4 Statistical Analysis
5.3.5 Computational Analysis of LEP Gene
5.4 Results
5.4.1 SNP Identification and Genotyping
5.4.2 Association Analysis with Growth Traits
5.4.3 Data Mining
5.4.4 Non-synonymous SNPs Functional Analysis for LEP
5.4.5 Mutant protein stability prediction for LEP
5.4.6 Structural Conformation and Conservation Analysis by Con Surf Server
5.4.7 LEP Protein Secondary Structure Prediction by PSIPRED
5.4.8 Homology Modelling
5.4.9 Ramachandran Plot Analysis
5.4.10 LEP Gene Protein-Protein Interaction
5.5 Discussion
5.6 Conclusion
CHAPTER6 General Conclusions,Innovations and Future Directions
6.1 Conclusions
6.2 Innovations
6.3 Future Directions
References
Acknowledgement
List of Publications
Appendices
本文编号：3747620
【文章页数】：210 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
ABBREVATIONS
CHAPTER1 Introduction and Literature Review
1.1 Introduction
1.1.1 Problem Statement
1.1.2 Justification
1.2 Candidate Gene Approach and Genetic Association
1.3 Genetic Markers
1.3.1 Restriction Fragment Length Polymorphisms(RFLPs)
1.3.2 Microsatellites
1.3.3 Single Nucleotide Polymorphisms
1.4 SNP Discovery Methods
1.5 Use of SNP Markers in Industry
1.6 The Sheep Growth and Development
1.7 Fat Deposition and Meat Value
1.8 The Melanocortin System Genes
1.9 Central Neural Circuits Energy Homeostasis
1.10 A Leptin/Melanocortin Pathway
1.11 MC4R Gene Polymorphism in Livestock
1.12 STAT3 Gene Polymorphism in Livestock
1.13 Overview of Experiments,Hypotheses and Objectives
1.13.1 General Overview
1.13.2 Study One:The Leptin-Melanocortin System Modulates Rapid Growth And Muscular High-Yield Carcass In Sheep Through Adipocytokine Signaling Pathway
1.13.3 Study Two:Genetic Variations in the MC4R Gene Linked To Elevated Body Weight and Fat-Related Traits in Sheep
1.13.4 Study Three:STAT3 Gene Expression and Its Role in Sheep Body Weight and Fatness Modulation
1.13.5 Study Four:Single Nucleotide Polymorphisms in the Leptin Gene and Their Associations with Growth and Fat-deposition Traits in the Sheep
CHAPTER2 The Leptin-Melanocortin System Modulates Rapid Growth and Muscular High-Yield Carcass in Sheep Through Adipocytokine Signaling Pathway
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1 Experimental Animals
2.3.2 Function and Pathway Enrichment Analysis
2.3.3 Tissue Collection and Transcriptome Data Analysis
2.3.4 Single Nucleotide Polymorphisms(SNPs)Detection from Re-Sequenced Data
2.3.5 Analyses of Positive Selection
2.4 Results
2.4.1 GO Function and KEGG Pathway Enrichment Analysis
2.4.2 Polymorphism Analysis
2.4.3 Positive Selection of the Leptin-Melanocortin Genes by FDIST Analysis
2.4.4 Analysis of Positive Selection
2.4.5 Haplotype Structure
2.4.6 The Relative Transcript Abundance of the Genes Involved In the Leptin-Melanocortin Signaling Pathway
2.5 Discussions
2.6 Conclusion
CHAPTER3 Genetic Variations in the MC4R Gene Linked To Elevated Body Weight and Fat-Related Traits in Sheep
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Ethical Statement
3.3.2 Experimental Animals and Measurements
3.3.3 Blood Collection
3.3.4 Isolation of Genomic DNA
3.3.5 Evaluation of the quality,purity,and concentration of DNA
3.3.6 Designing of Primers
3.3.7 PCR Reaction Mixture
3.3.8 PCR Program
3.3.9 Checking of Amplified PCR Products
3.3.10 Identification of Single Nucleotide Polymorphisms(SNPs)
3.3.11 RNA Extraction and Real-Time q PCR
3.3.12 Sequence Characterization of the Potential Promoter Region of MC4R in Sheep
3.3.13 Promoter Cloning and Generation of Luciferase Reporter Constructs
3.3.14 Purification of PCR product by PCR Clean-Up System(Omega Bio-Tek,Inc.,Norcross,GA,USA)
3.3.15 Vector Ligation of Amplified Products
3.3.16 Competent Cell Preparation and Transformation
3.3.17 Confirmation of Positive Clones
3.3.18 Colony PCR
3.3.19 Plasmid Isolation from Positive Clones
3.3.20 Plasmid Digestion by Restriction Endonuclease Enzymes
3.3.21 Cell Culture,Transfection,and Luciferase Assays
3.3.22 Statistical Analysis
3.3.23 Association Analysis
3.3.24 Expression Analysis
3.4 Results
3.4.1 Single Nucleotide Polymorphism(SNP)Identification
3.4.2 SNP Variation in Potential Cis-Regulatory Elements of the MC4R Gene
3.4.3 Association Analyses
3.4.4 Linkage disequilibrium and Haplotype Analysis of the MC4R Gene
3.4.5 Sequence Analysis of the Promoter Region of MC4R Gene
3.4.6 Differential Expression of the MC4R Gene across Age and Sex
3.4.7 The Proximal Minimal Promoter Region of the MC4R Gene
3.5 Discussion
3.6 Conclusions
CHAPTER4 STAT3 Gene Expression and Its Role in Sheep Body Weight and Fatness Modulation
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.3.1 Sample Collection
4.3.2 Lipid Extraction-Soxhlet Method
4.3.3 PCR Amplification,Sequencing,and Genotyping
4.3.4 Sequence Analysis
4.3.5 Collection of RNA Samples
4.3.6 RNA Purification and c DNA Synthesis
4.3.7 RT-PCR Analysis of Expression Patterns
4.3.8 Immunohistochemical Localization and Analysis of STAT3
4.3.9 Statistical analysis
4.4 Results
4.4.1 The sequence of the sheep STAT3 gene
4.4.2 STAT3 Relative m RNA Expression in Sheep
4.4.3 Immunohistochemical Analysis
4.4.4 SNP Identification and Analysis of Its Association with Body Size Traits
4.4.5 Effect of the Polymorphisms on STAT3 Locus on Body Measurement and Fat Deposition Traits
4.4.6 Linkage Disequilibrium and Haplotype Analysis
4.5 Discussion
4.6 Conclusions
CHAPTER5 Single Nucleotide Polymorphisms in the LEP Gene and Their Associations with Growth and Fat-deposition Traits in the Sheep
5.1 Abstract
5.2 Introductions
5.3 Experimental Section
5.3.1 Materials and Phenotypic Data Collection
5.3.2 PCR Amplification and SNP Identification
5.3.3 SNP Identification and Genotyping
5.3.4 Statistical Analysis
5.3.5 Computational Analysis of LEP Gene
5.4 Results
5.4.1 SNP Identification and Genotyping
5.4.2 Association Analysis with Growth Traits
5.4.3 Data Mining
5.4.4 Non-synonymous SNPs Functional Analysis for LEP
5.4.5 Mutant protein stability prediction for LEP
5.4.6 Structural Conformation and Conservation Analysis by Con Surf Server
5.4.7 LEP Protein Secondary Structure Prediction by PSIPRED
5.4.8 Homology Modelling
5.4.9 Ramachandran Plot Analysis
5.4.10 LEP Gene Protein-Protein Interaction
5.5 Discussion
5.6 Conclusion
CHAPTER6 General Conclusions,Innovations and Future Directions
6.1 Conclusions
6.2 Innovations
6.3 Future Directions
References
Acknowledgement
List of Publications
Appendices
本文编号：3747620
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