Functional Characterization of NF-YB1,NF-YC12 and bHLH144 in
发布时间：2024-06-02 04:35
鉴定种子发育过程中的重要调控基因的鉴定是水稻稻米品质遗传改良的关键。含有NF-YA,B和C亚基的核因子Y(NF-Y)异源三聚体是真核生物中重要的转录调节因子。前人研究表明一些主要在种子中表达的NF-Y核因子是水稻种子发育过程中的主要调节因子,但其具体分子机制仍不清楚。在本研究中,我们报导了水稻NF-Y异源三聚体NF-YB1-YC12-bHLH144的功能特征,由酵母三杂和体外Pull down实验确定了该异源三聚体的结合顺序,即NF-YB1先结合NF-YC12,然后再结合bHLH144。通过CRISPR/Cas9分别敲除该复合体的每个成员均导致稻米品质的改变,同时nf-yb1和nfyc12突变体的粒型变小。RNA-seq分析鉴定到由NF-YB1和NF-YC12共同调节的1496个基因,包括关键的颗粒结合淀粉合成酶基因Wx。降解实验结果表明,NF-YC12和bHLH144可以抑制NF-YB1受泛素/26S蛋白酶体介导的降解,并且凝胶阻滞实验(EMSA),染色质免疫共沉淀(ChIP)-PCR和瞬时荧光素酶活性检测等实验表明NF-YB1可直接与Wx基因启动子上的“G-box”基序结合并激活其...
【文章页数】：97 页
【学位级别】：博士
【文章目录】：
List of abbreviations and acronyms
中文摘要
abstract
CHAPTER One Introduction
1.1 Nuclear factor Y (NF-Y) transcription factors
1.2 Classification of rice NF-Y genes
1.3 Literature review
1.4 bZIP proteins
CHAPTER Two Functional characterization of NF-YB1,NF-YC12 and bHLH144 in rice(Oryza sativa L.)
2.1 Materials and methods
2.1.1 Plant growth conditions and phenotypical characterizations
2.1.2 RNA isolation, qRT-PCR and mRNA in-situ hybridization
2.1.3 Vector construction and plant transformation
2.1.4 Bacterial-two-hybrid assay
2.1.5 Purification of tag-fused proteins and in vitro pull-down assay
2.1.6 Yeast-three-hybrid assay
2.1.7 BIFC assay
2.1.8 Yeast-one-hybrid assay
2.1.9 Luciferase transient transcriptional activity assay
2.1.10 Electrophoresis Mobility Shift Assay
2.1.11 Chromatin immuno-precipitation (ChIP) and ChIP-PCR
2.1.12 Cell-free degradation Assay
2.2 Results
2.2.1 NF-YB 1 is a seed-specific gene and its protein locates in nuclear and cytoplasm
2.2.2 Knock-down of NF-YB1 reduced grain size and altered grain quality
2.2.3 NF-YB1,NF-YC12 and bHLH144 forms a hereotrimer complex in a sequential order
2.2.4 cmf-ycl2 and crbhlhl44 exhibited similar phenotype to crnf-yb1 in seed development
2.2.5 NF-YB1 and NF-YC12co-regulate transcription of genes involves in starch biosynthess
2.2.6 NF-YB1 binds to the G-box of Wx promoter to activate its transcription
2.2.7 NF-YC12 and bHLH144 enhances NF-YB1 stability
2.3 Discussion
2.3.1 Comprehensive effects of NF-YB 1 on seed development
2.3.2 NF-YB directly activates Wxvia binding to the G-box in promoter
2.3.3 NF-YB1-YC12-bHLH144 works as a complex
2.4 Conclusion
CHAPTER Three The regulatory mechanism of OsRISBZ1, a basic leucine zippertranscription factor involved in rice seed development
3.1 Materials and methods
3.1.1 Plant growth conditions and phenotypical characterizations
3.1.2 RNA isolation andqRT-PCR
3.1.3 Vector construction and plant transformation
3.1.4 Purification of tag-fused proteins and in vitro pull-down assay
3.1.5 In vitro Kinase Assay
3.1.6 Luciferase transient transcriptional activity assay
3.1.7 Electrophoresis Mobility Shift Assay
3.2 Results
3.2.1 Expression patterns of RISBZ1 in different tissue and phenotypical characterization of risbzl and WT
3.2.2 RISBZ1 interact physically with SAPK10
3.2.3 RISBZlbinds to the G-box of Wx promoter to activate its transcription
3.3 Discussion
3.4 Perspective and future research
References
Supporting information
Acknowledgement
List of publication during postdoctoral
Permanent home address
本文编号：3986862
【文章页数】：97 页
【学位级别】：博士
【文章目录】：
List of abbreviations and acronyms
中文摘要
abstract
CHAPTER One Introduction
1.1 Nuclear factor Y (NF-Y) transcription factors
1.2 Classification of rice NF-Y genes
1.3 Literature review
1.4 bZIP proteins
CHAPTER Two Functional characterization of NF-YB1,NF-YC12 and bHLH144 in rice(Oryza sativa L.)
2.1 Materials and methods
2.1.1 Plant growth conditions and phenotypical characterizations
2.1.2 RNA isolation, qRT-PCR and mRNA in-situ hybridization
2.1.3 Vector construction and plant transformation
2.1.4 Bacterial-two-hybrid assay
2.1.5 Purification of tag-fused proteins and in vitro pull-down assay
2.1.6 Yeast-three-hybrid assay
2.1.7 BIFC assay
2.1.8 Yeast-one-hybrid assay
2.1.9 Luciferase transient transcriptional activity assay
2.1.10 Electrophoresis Mobility Shift Assay
2.1.11 Chromatin immuno-precipitation (ChIP) and ChIP-PCR
2.1.12 Cell-free degradation Assay
2.2 Results
2.2.1 NF-YB 1 is a seed-specific gene and its protein locates in nuclear and cytoplasm
2.2.2 Knock-down of NF-YB1 reduced grain size and altered grain quality
2.2.3 NF-YB1,NF-YC12 and bHLH144 forms a hereotrimer complex in a sequential order
2.2.4 cmf-ycl2 and crbhlhl44 exhibited similar phenotype to crnf-yb1 in seed development
2.2.5 NF-YB1 and NF-YC12co-regulate transcription of genes involves in starch biosynthess
2.2.6 NF-YB1 binds to the G-box of Wx promoter to activate its transcription
2.2.7 NF-YC12 and bHLH144 enhances NF-YB1 stability
2.3 Discussion
2.3.1 Comprehensive effects of NF-YB 1 on seed development
2.3.2 NF-YB directly activates Wxvia binding to the G-box in promoter
2.3.3 NF-YB1-YC12-bHLH144 works as a complex
2.4 Conclusion
CHAPTER Three The regulatory mechanism of OsRISBZ1, a basic leucine zippertranscription factor involved in rice seed development
3.1 Materials and methods
3.1.1 Plant growth conditions and phenotypical characterizations
3.1.2 RNA isolation andqRT-PCR
3.1.3 Vector construction and plant transformation
3.1.4 Purification of tag-fused proteins and in vitro pull-down assay
3.1.5 In vitro Kinase Assay
3.1.6 Luciferase transient transcriptional activity assay
3.1.7 Electrophoresis Mobility Shift Assay
3.2 Results
3.2.1 Expression patterns of RISBZ1 in different tissue and phenotypical characterization of risbzl and WT
3.2.2 RISBZ1 interact physically with SAPK10
3.2.3 RISBZlbinds to the G-box of Wx promoter to activate its transcription
3.3 Discussion
3.4 Perspective and future research
References
Supporting information
Acknowledgement
List of publication during postdoctoral
Permanent home address
本文编号：3986862
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