瘦素-黑皮素信號(hào)通路相關(guān)基因的變異及其對(duì)綿羊體尺和脂肪厚度性狀的影響
發(fā)布時(shí)間：2023-02-21 13:55
綿羊是重要的畜種,它能夠?yàn)槲覀兲峁┭蛉�、羊奶、羊皮和羊毛等多種產(chǎn)品。對(duì)于肉用綿羊,生長(zhǎng)和繁殖是選育的主要目標(biāo)。但是,與綿羊生長(zhǎng)和產(chǎn)肉相關(guān)的遺傳因素還沒有完全確認(rèn)。因此,研究調(diào)節(jié)控制生長(zhǎng)和產(chǎn)肉性狀的代謝通路和基因?qū)樘岣哐蛉猱a(chǎn)量和質(zhì)量提供新策略。瘦素-黑皮質(zhì)素信號(hào)通路上的基因在體重調(diào)節(jié)和能量穩(wěn)態(tài)中至關(guān)重要。該通路上的相關(guān)基因,尤其是黑皮質(zhì)素4受體(MC4R)、信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3(STAT3)和瘦蛋白(LEP)基因能夠通過影響下丘腦的活動(dòng)來調(diào)控?cái)z食和能量消耗�；谠谌祟惡推渌锓N中的相關(guān)報(bào)道的啟發(fā),本研究認(rèn)為很有必要確定瘦素-黑皮質(zhì)素信號(hào)通路相關(guān)基因上的變異和這些變異對(duì)綿羊體尺和體脂性狀的影響,特別是闡明這些基因的結(jié)構(gòu)和功能以及它們?cè)诰d羊不同組織中的表達(dá)量、鑒定那些位于MC4R基因啟動(dòng)子區(qū)上對(duì)該基因的轉(zhuǎn)錄調(diào)控起到重要影響的變異。在本研究中,我們選擇了5個(gè)中國地方綿羊品種,總計(jì)551只綿羊作為樣本。使用聚合酶鏈反應(yīng)(PCR)直接測(cè)序方法檢測(cè)了這些基因的變異。用一般線性模型(GLM)研究這些變異與各種體尺和體脂性狀間的關(guān)聯(lián)。我們收集了42只綿羊從出生到六月齡的七個(gè)組織(下丘腦,腎,肝,心...
【文章頁數(shù)】：210 頁
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：3747620
【文章頁數(shù)】：210 頁
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：3747620
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