TORCs在秦川肉牛脂肪细胞增殖和分化中的基因功能及转录调控分析
发布时间：2021-06-28 18:18
肌内脂肪组织（大理石花纹）含量较低在中国仍然是提高牛肉产品质量的挑战,大理石花纹含量高的牛肉更受欢迎。因此,增加IMF含量的方法已成为改善肉质的关键方面。因此,对脂肪形成机理的研究为改善肉质提供了宝贵的信息。本研究探究了TORCs及其对牛脂肪细胞脂质代谢的潜在调控机制。TORC基因家族共有三个成员:TORC1,TORC2和TORC3,也称为CRTC[CREB（c AMP反应元件结合蛋白）调节的转录共激活因子]。CREB转录需要TORC,CREB,是一种通用的转录因子,可调节4000多个基因的表达,参与代谢,细胞增殖,分化,免疫应答以及其他生理和病理过程的调控。TORC2基因通过PI3K-Akt,AMPK,胰高血糖素和胰岛素抵抗等信号通路调控营养代谢,糖异生,肌异生和脂肪形成。在本研究中,我们探索了牛脂肪细胞中TORC2基因的功能。我们还通过不同的实验探索了TORC1和TORC2基因的转录和转录后调控,并证实了以下发现。1.PCR扩增产物测序在秦川牛的TORC2基因启动子区域中分别在g.16534694G>A,g.16535011C>T和g.16535044A>T等位点...
【文章来源】：西北农林科技大学陕西省 211工程院校 985工程院校 教育部直属院校
【文章页数】：169 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
Chapter1 Review of literature
1.1 Purpose and significance of the Research
1.2 Meat Quality
1.3 Transcriptional Regulation of Adipogenic Marker genes in Bovine Species
1.4 The role of KLF6 and PU.1 in the regulation of Bovine ELOVL6
1.5 Roles of Evi1 and C/EBPαin the transcriptional regulation of bovine ABHD5 gene in Preadipocytes of Qinchuan Cattle
1.6 The role of KLF15 in transcriptional regulation of KLF3 gene in bovine adipocyte
1.7 Roles of E2F1,PLAG1,C/EBPβ,and SMAD3 in the regulation of perilipin1 (PLIN1)in bovine adipocytes
1.8 Post Transcriptional Regulation of Adipogenic Marker genes
Chapter2 Genetic variants in the TORC2 gene promoter and their association with body measurement and carcass quality traits in Qinchuan cattle
2.1 Materials and Methods
2.1.1 Ethical statement
2.1.2 Phenotypic data and DNA sample collection
2.1.3 PCR amplification and genotyping
2.1.4 Potential cis-acting element identification
2.1.5 Construction of Plasmid,Isolation,Culture and transfection of preadipocyte cells for luciferase reporter assay
2.1.6 Estimates of conservation and biological evolution
2.1.7 Tissue collection,RNA extraction,preparation of c DNA and real-time PCR
2.1.8 Data Analyses
2.2 Results
2.2.1 SNP identification
2.2.2 Linkage disequilibrium and haplotype identification of the bovine TORC2 gene
2.2.3 Association of genotype and diplotype with physical measurements and carcass quality traits
2.2.4 Transcription factor binding site prediction
2.2.5 Luciferase reporter assay
2.2.6 Bioinformatics study of the TORC2 gene
2.2.7 Relative m RNA expression of the TORC2 gene at different ages
2.3 Discussion
Chapter3.Function and Transcriptional Regulation of Bovine TORC2 Gene in Adipocytes:Roles of C/EBP?,XBP1,INSM1 and ZNF
3.1 Materials and Methods
3.1.1 Ethics Statement
3.1.2 Tissue Collection and m RNA Expression
3.1.3 Isolation of Bovine Primary Preadipocytes
3.1.4 Cell Culture and Immunofluorescence
3.1.5 Ed U Proliferation Assay
3.1.6 Cell Cycle Assay through Flow Cytometry
3.1.7 CCK-8 Assay
3.1.8 5′-Rapid Amplification of c DNA Ends(RACE)
3.1.9 DNA Extraction and Amplification of TORC2 Gene Promoter
3.1.10 Cloning of TORC2 Gene Promoter
3.1.11 Cell Culture and Transient Transfection
3.1.12 Mutagenesis in Transcription Factor Binding Sites
3.1.13 C/BEP?,XBP1,ZNF263 and INSM1 Knockdown
3.1.14 Western Blot Analysis
3.1.15 Cell Differentiation and Oil Red O Staining
3.1.16 EMSAs(Electrophoretic Mobility Shift Assays)
3.1.17 Statistical Analysis
3.2 Results
3.2.1 Transfection Efficiency,Tissues and Cellular Expression of TORC2 Gene
3.2.2 TORC2 promotes preadipocyte proliferation
3.2.3 TORC2 Enhance Adipocyte Differentiation
3.2.4 Identification of Transcription Start Site(TSS)of the TORC2 Gene
3.2.5 Identification of Core Promoter Region of TORC2 Gene
3.2.6 Roles of C/EBP?,XBP1,INSM1 and ZNF263 in Transcriptional Regulation of TORC2 Gene
3.2.7 Genetic Interaction with Transcription Factors
3.2.8 Silencing of C/EBP?,ZNF263,XBP1 and INSM1 Transcription Factors
3.2.9 Oil Red O Staining
3.2.10 DNA-Protein Interaction through EMSAs
3.3 Discussion
3.4 Conclusion
Chapter4.RNA-Seq Reveal Role of Bovine TORC2 in the Regulation of Adipogenesis
4.1.Materials and Methods
4.1.1 Ethical Statement
4.1.2 Isolation of Bovine Primary Preadipocytes
4.1.3 Cell Differentiation and Oil Red O Staining
4.1.4 RNA isolation,c DNA library,and q RT-PCR
4.1.5 Western Blot Analysis
4.1.6 Construction of RNA-Seq library,quality control and sequencing
4.1.7 DEG identification
4.1.8 Functional Enrichment Analysis
4.1.9 Statistical Analysis
4.2 Results
4.2.1 Expression,down-regulation of TORC2 gene,and quality evaluation of the samples
4.2.2 Role of TORC2 in adipogenesis through deep sequencing analysis
4.2.3 Role of TORC2 in transcriptional regulation of genes responsible for cellular differentiation
4.2.4 Identification and Validation of the DEGs during Adipogenesis Based on q RT-PCR
4.3 Discussion
4.4 Conclusion
Chapter5 Bioinformatics analysis and transcription regulation of TORC1 gene through transcription factors NRF1 and Smad3 in bovine preadipocytes
5.1 Materials& Methods
5.1.1 Ethical Statement
5.1.2 Bioinformatics study
5.1.3 Tissue Collection and m RNA Expression
5.1.4 Cell Culture and Immunofluorescence
5.1.5 DNA Extraction and Amplification of TORC1 Gene Promoter
5.1.6 Cloning of TORC1 Gene Promoter and Luciferase Reporter Assay
5.1.7 Isolation,Cell Culture and Transient Transfection of Bovine Primary Preadipocytes
5.1.8 Western Blot Analysis
5.1.9 Mutagenesis in Transcription Factor Binding Sites
5.1.10 NRF1 and SMAD3 Knockdown
5.1.11 EMSA(Electrophoretic Mobility Shift Assays)
5.1.12 Statistical Analysis
5.2 Results
5.2.1 Biological Evolution and Estimates of Conservation
5.2.2 Tissues and Cellular Expression of TORC1 Gene
5.2.3 Identification of Minimum Proximal Promoter of TORC1 Gene
5.2.4 Roles of NRF1 and Smad3 in Transcriptional Regulation of TORC1 Gene
5.2.5 Genetic Interaction with Transcription Factors
5.2.6 Validation of the roles of NRF1 and Smad3 TFs in the transcriptional regulation of TORC1 gene
5.2.7 Silencing of NRF1 and Smad3 Transcription Factors
5.2.8 DNA-Protein interaction through EMSAs
5.3 Discussion
5.4 Conclusion
Chapter6 Bta-mi R-149-5p Inhibits Proliferation and Differentiation of Bovine Adipocytes through Targeting TORCs at Both Transcriptional and Post Transcriptional Levels
6.1 Materials and Methods
6.1.1 Ethics Statement
6.1.2 Isolation of Bovine Primary Preadipocytes
6.1.3 Cell Culture and Transient Transfection
6.1.4 Extraction of RNA,Construction of the c DNA Library and q PCR Analysis
6.1.5 Western Blot Analysis
6.1.6 Cell Differentiation and Oil Red O Staining
6.1.7 Immunocytochemical Analysis
6.1.8 Ed U Proliferation Assay
6.1.9 Cell Cycle Assay through Flow Cytometry
6.1.10 CCK-8 Assay
6.1.11 Luciferase Activity Assay
6.1.12 Statistical Analysis
6.2 Results
6.2.1 Expression Profile of bta-mi R-149-5p During Adipogenesis
6.2.2 Bta-mi R-149-5p Inhibits Preadipocyte Proliferation
6.2.3 Bta-mi R-149-5p Regulate Adipogenesis through Directly Targeting TORC2 and TORC1 Genes
6.2.4 Bta-mi R-149-5p Regulate TORC2 and TORC1 through Transcriptional Reregulation of TFs Present in Their Core Promoter
6.2.5 Bta-mi R-149-5p Represses Adipocyte Differentiation
6.3 Discussion
6.4 Conclusion
Thesis conclusion
Acknowledgment
References
Appendices
Appendix of Chapter 2
Appendix of Chapter 3
Appendix of Chapter 4
Appendix of Chapter 5
Appendix of Chapter 6
About the Author
【参考文献】：
期刊论文
[1]秦川牛IGF2基因SNPs检测及其与胴体、肉质性状的相关性[J]. 韩瑞华,昝林森,杨大鹏,郝荣超. 遗传. 2008(12)
本文编号：3254811
【文章来源】：西北农林科技大学陕西省 211工程院校 985工程院校 教育部直属院校
【文章页数】：169 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
Chapter1 Review of literature
1.1 Purpose and significance of the Research
1.2 Meat Quality
1.3 Transcriptional Regulation of Adipogenic Marker genes in Bovine Species
1.4 The role of KLF6 and PU.1 in the regulation of Bovine ELOVL6
1.5 Roles of Evi1 and C/EBPαin the transcriptional regulation of bovine ABHD5 gene in Preadipocytes of Qinchuan Cattle
1.6 The role of KLF15 in transcriptional regulation of KLF3 gene in bovine adipocyte
1.7 Roles of E2F1,PLAG1,C/EBPβ,and SMAD3 in the regulation of perilipin1 (PLIN1)in bovine adipocytes
1.8 Post Transcriptional Regulation of Adipogenic Marker genes
Chapter2 Genetic variants in the TORC2 gene promoter and their association with body measurement and carcass quality traits in Qinchuan cattle
2.1 Materials and Methods
2.1.1 Ethical statement
2.1.2 Phenotypic data and DNA sample collection
2.1.3 PCR amplification and genotyping
2.1.4 Potential cis-acting element identification
2.1.5 Construction of Plasmid,Isolation,Culture and transfection of preadipocyte cells for luciferase reporter assay
2.1.6 Estimates of conservation and biological evolution
2.1.7 Tissue collection,RNA extraction,preparation of c DNA and real-time PCR
2.1.8 Data Analyses
2.2 Results
2.2.1 SNP identification
2.2.2 Linkage disequilibrium and haplotype identification of the bovine TORC2 gene
2.2.3 Association of genotype and diplotype with physical measurements and carcass quality traits
2.2.4 Transcription factor binding site prediction
2.2.5 Luciferase reporter assay
2.2.6 Bioinformatics study of the TORC2 gene
2.2.7 Relative m RNA expression of the TORC2 gene at different ages
2.3 Discussion
Chapter3.Function and Transcriptional Regulation of Bovine TORC2 Gene in Adipocytes:Roles of C/EBP?,XBP1,INSM1 and ZNF
3.1 Materials and Methods
3.1.1 Ethics Statement
3.1.2 Tissue Collection and m RNA Expression
3.1.3 Isolation of Bovine Primary Preadipocytes
3.1.4 Cell Culture and Immunofluorescence
3.1.5 Ed U Proliferation Assay
3.1.6 Cell Cycle Assay through Flow Cytometry
3.1.7 CCK-8 Assay
3.1.8 5′-Rapid Amplification of c DNA Ends(RACE)
3.1.9 DNA Extraction and Amplification of TORC2 Gene Promoter
3.1.10 Cloning of TORC2 Gene Promoter
3.1.11 Cell Culture and Transient Transfection
3.1.12 Mutagenesis in Transcription Factor Binding Sites
3.1.13 C/BEP?,XBP1,ZNF263 and INSM1 Knockdown
3.1.14 Western Blot Analysis
3.1.15 Cell Differentiation and Oil Red O Staining
3.1.16 EMSAs(Electrophoretic Mobility Shift Assays)
3.1.17 Statistical Analysis
3.2 Results
3.2.1 Transfection Efficiency,Tissues and Cellular Expression of TORC2 Gene
3.2.2 TORC2 promotes preadipocyte proliferation
3.2.3 TORC2 Enhance Adipocyte Differentiation
3.2.4 Identification of Transcription Start Site(TSS)of the TORC2 Gene
3.2.5 Identification of Core Promoter Region of TORC2 Gene
3.2.6 Roles of C/EBP?,XBP1,INSM1 and ZNF263 in Transcriptional Regulation of TORC2 Gene
3.2.7 Genetic Interaction with Transcription Factors
3.2.8 Silencing of C/EBP?,ZNF263,XBP1 and INSM1 Transcription Factors
3.2.9 Oil Red O Staining
3.2.10 DNA-Protein Interaction through EMSAs
3.3 Discussion
3.4 Conclusion
Chapter4.RNA-Seq Reveal Role of Bovine TORC2 in the Regulation of Adipogenesis
4.1.Materials and Methods
4.1.1 Ethical Statement
4.1.2 Isolation of Bovine Primary Preadipocytes
4.1.3 Cell Differentiation and Oil Red O Staining
4.1.4 RNA isolation,c DNA library,and q RT-PCR
4.1.5 Western Blot Analysis
4.1.6 Construction of RNA-Seq library,quality control and sequencing
4.1.7 DEG identification
4.1.8 Functional Enrichment Analysis
4.1.9 Statistical Analysis
4.2 Results
4.2.1 Expression,down-regulation of TORC2 gene,and quality evaluation of the samples
4.2.2 Role of TORC2 in adipogenesis through deep sequencing analysis
4.2.3 Role of TORC2 in transcriptional regulation of genes responsible for cellular differentiation
4.2.4 Identification and Validation of the DEGs during Adipogenesis Based on q RT-PCR
4.3 Discussion
4.4 Conclusion
Chapter5 Bioinformatics analysis and transcription regulation of TORC1 gene through transcription factors NRF1 and Smad3 in bovine preadipocytes
5.1 Materials& Methods
5.1.1 Ethical Statement
5.1.2 Bioinformatics study
5.1.3 Tissue Collection and m RNA Expression
5.1.4 Cell Culture and Immunofluorescence
5.1.5 DNA Extraction and Amplification of TORC1 Gene Promoter
5.1.6 Cloning of TORC1 Gene Promoter and Luciferase Reporter Assay
5.1.7 Isolation,Cell Culture and Transient Transfection of Bovine Primary Preadipocytes
5.1.8 Western Blot Analysis
5.1.9 Mutagenesis in Transcription Factor Binding Sites
5.1.10 NRF1 and SMAD3 Knockdown
5.1.11 EMSA(Electrophoretic Mobility Shift Assays)
5.1.12 Statistical Analysis
5.2 Results
5.2.1 Biological Evolution and Estimates of Conservation
5.2.2 Tissues and Cellular Expression of TORC1 Gene
5.2.3 Identification of Minimum Proximal Promoter of TORC1 Gene
5.2.4 Roles of NRF1 and Smad3 in Transcriptional Regulation of TORC1 Gene
5.2.5 Genetic Interaction with Transcription Factors
5.2.6 Validation of the roles of NRF1 and Smad3 TFs in the transcriptional regulation of TORC1 gene
5.2.7 Silencing of NRF1 and Smad3 Transcription Factors
5.2.8 DNA-Protein interaction through EMSAs
5.3 Discussion
5.4 Conclusion
Chapter6 Bta-mi R-149-5p Inhibits Proliferation and Differentiation of Bovine Adipocytes through Targeting TORCs at Both Transcriptional and Post Transcriptional Levels
6.1 Materials and Methods
6.1.1 Ethics Statement
6.1.2 Isolation of Bovine Primary Preadipocytes
6.1.3 Cell Culture and Transient Transfection
6.1.4 Extraction of RNA,Construction of the c DNA Library and q PCR Analysis
6.1.5 Western Blot Analysis
6.1.6 Cell Differentiation and Oil Red O Staining
6.1.7 Immunocytochemical Analysis
6.1.8 Ed U Proliferation Assay
6.1.9 Cell Cycle Assay through Flow Cytometry
6.1.10 CCK-8 Assay
6.1.11 Luciferase Activity Assay
6.1.12 Statistical Analysis
6.2 Results
6.2.1 Expression Profile of bta-mi R-149-5p During Adipogenesis
6.2.2 Bta-mi R-149-5p Inhibits Preadipocyte Proliferation
6.2.3 Bta-mi R-149-5p Regulate Adipogenesis through Directly Targeting TORC2 and TORC1 Genes
6.2.4 Bta-mi R-149-5p Regulate TORC2 and TORC1 through Transcriptional Reregulation of TFs Present in Their Core Promoter
6.2.5 Bta-mi R-149-5p Represses Adipocyte Differentiation
6.3 Discussion
6.4 Conclusion
Thesis conclusion
Acknowledgment
References
Appendices
Appendix of Chapter 2
Appendix of Chapter 3
Appendix of Chapter 4
Appendix of Chapter 5
Appendix of Chapter 6
About the Author
【参考文献】：
期刊论文
[1]秦川牛IGF2基因SNPs检测及其与胴体、肉质性状的相关性[J]. 韩瑞华,昝林森,杨大鹏,郝荣超. 遗传. 2008(12)
本文编号：3254811
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