TORCs在秦川肉牛脂肪細(xì)胞增殖和分化中的基因功能及轉(zhuǎn)錄調(diào)控分析
發(fā)布時(shí)間：2021-06-28 18:18
肌內(nèi)脂肪組織（大理石花紋）含量較低在中國(guó)仍然是提高牛肉產(chǎn)品質(zhì)量的挑戰(zhàn),大理石花紋含量高的牛肉更受歡迎。因此,增加IMF含量的方法已成為改善肉質(zhì)的關(guān)鍵方面。因此,對(duì)脂肪形成機(jī)理的研究為改善肉質(zhì)提供了寶貴的信息。本研究探究了TORCs及其對(duì)牛脂肪細(xì)胞脂質(zhì)代謝的潛在調(diào)控機(jī)制。TORC基因家族共有三個(gè)成員:TORC1,TORC2和TORC3,也稱為CRTC[CREB（c AMP反應(yīng)元件結(jié)合蛋白）調(diào)節(jié)的轉(zhuǎn)錄共激活因子]。CREB轉(zhuǎn)錄需要TORC,CREB,是一種通用的轉(zhuǎn)錄因子,可調(diào)節(jié)4000多個(gè)基因的表達(dá),參與代謝,細(xì)胞增殖,分化,免疫應(yīng)答以及其他生理和病理過(guò)程的調(diào)控。TORC2基因通過(guò)PI3K-Akt,AMPK,胰高血糖素和胰島素抵抗等信號(hào)通路調(diào)控營(yíng)養(yǎng)代謝,糖異生,肌異生和脂肪形成。在本研究中,我們探索了牛脂肪細(xì)胞中TORC2基因的功能。我們還通過(guò)不同的實(shí)驗(yàn)探索了TORC1和TORC2基因的轉(zhuǎn)錄和轉(zhuǎn)錄后調(diào)控,并證實(shí)了以下發(fā)現(xiàn)。1.PCR擴(kuò)增產(chǎn)物測(cè)序在秦川牛的TORC2基因啟動(dòng)子區(qū)域中分別在g.16534694G>A,g.16535011C>T和g.16535044A>T等位點(diǎn)...
【文章來(lái)源】：西北農(nóng)林科技大學(xué)陜西省 211工程院校 985工程院校 教育部直屬院校
【文章頁(yè)數(shù)】：169 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]秦川牛IGF2基因SNPs檢測(cè)及其與胴體、肉質(zhì)性狀的相關(guān)性[J]. 韓瑞華,昝林森,楊大鵬,郝榮超. 遺傳. 2008(12)
本文編號(hào)：3254811
【文章來(lái)源】：西北農(nóng)林科技大學(xué)陜西省 211工程院校 985工程院校 教育部直屬院校
【文章頁(yè)數(shù)】：169 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]秦川牛IGF2基因SNPs檢測(cè)及其與胴體、肉質(zhì)性狀的相關(guān)性[J]. 韓瑞華,昝林森,楊大鵬,郝榮超. 遺傳. 2008(12)
本文編號(hào)：3254811
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