Functional Characterization of NF-YB1,NF-YC12 and bHLH144 in
發(fā)布時間：2024-06-02 04:35
鑒定種子發(fā)育過程中的重要調(diào)控基因的鑒定是水稻稻米品質(zhì)遺傳改良的關(guān)鍵。含有NF-YA,B和C亞基的核因子Y(NF-Y)異源三聚體是真核生物中重要的轉(zhuǎn)錄調(diào)節(jié)因子。前人研究表明一些主要在種子中表達(dá)的NF-Y核因子是水稻種子發(fā)育過程中的主要調(diào)節(jié)因子,但其具體分子機(jī)制仍不清楚。在本研究中,我們報導(dǎo)了水稻NF-Y異源三聚體NF-YB1-YC12-bHLH144的功能特征,由酵母三雜和體外Pull down實驗確定了該異源三聚體的結(jié)合順序,即NF-YB1先結(jié)合NF-YC12,然后再結(jié)合bHLH144。通過CRISPR/Cas9分別敲除該復(fù)合體的每個成員均導(dǎo)致稻米品質(zhì)的改變,同時nf-yb1和nfyc12突變體的粒型變小。RNA-seq分析鑒定到由NF-YB1和NF-YC12共同調(diào)節(jié)的1496個基因,包括關(guān)鍵的顆粒結(jié)合淀粉合成酶基因Wx。降解實驗結(jié)果表明,NF-YC12和bHLH144可以抑制NF-YB1受泛素/26S蛋白酶體介導(dǎo)的降解,并且凝膠阻滯實驗(EMSA),染色質(zhì)免疫共沉淀(ChIP)-PCR和瞬時熒光素酶活性檢測等實驗表明NF-YB1可直接與Wx基因啟動子上的“G-box”基序結(jié)合并激活其...
【文章頁數(shù)】：97 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：3986862
【文章頁數(shù)】：97 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：3986862
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