水稻类根瘤起始蛋白OsNLP1和拟南芥AtNLP7在作物氮利用效率方面的功能解析
发布时间：2021-11-24 15:43
氮（N）是植物正常生长发育和农业生产所必需的一种大量元素。农民在农业生产中施用大量的氮肥以维持作物的高产。但是,由于氮肥利用效率过低,大部分没有被植物吸收的氮肥流失到环境中,造成严重的环境污染并且大大增加了农业生产的成本。解决这些问题的一个理想且经济的解决方案是提高作物的氮利用效率,这一直是全世界科学家研究的焦点,但关于它的遗传决定因素和调控机理还有待进一步的探究。水稻是世界上最重要的粮食作物之一,为30亿人提供35%-60%的热量。提高水稻的氮利用效率是很有必要的。我们的工作表明了水稻NIN-LIKE PROTEIN 1（OsNLP1）在提高NUE过程中起着核心的作用。OsNLP1蛋白定位于细胞核,N饥饿处理可以在转录水平迅速的诱导其表达。在不同施氮条件下,过量表达OsNLP1都提高了水稻的生长、籽粒产量和NUE,而敲除OsNLP1则降低了低氮条件下的籽粒产量和NUE。OsNLP1通过协同调控多个氮素吸收和同化的基因来调控硝态氮和铵态氮的利用。染色质免疫沉淀和酵母单杂交实验分析表明,OsNLP1可以直接结合这些基因的启动子来激活它们的表达。因此,我们的实验结果表明OsNLP1是氮素利...
【文章来源】：中国科学技术大学安徽省 211工程院校 985工程院校
【文章页数】：118 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Chapter 1 Introduction
1.1 The importance of nitrogen in plant growth and agricultural production
1.2 Plant N use efficiency
1.3 Plant N absorption and metabolic pathways
1.3.1 Nitrate nitrogen absorption and metabolism
1.3.2 Ammonium nitrogen absorption and metabolism
1.3.3 N and carbon metabolism balance
1.3.4 Research status of nitrate signaling in plants
1.4 Introduction of plant RWP-RK transcription factor family
1.4.1 Relationship and typical structural features of thresholds in RWP-RK family system
1.4.2 The Function of RWP-RK protein
1.5 The Function of NLPs in rice and cotton crops
Chapter 2 Experimental Materials and Methods
2.1 Plant materials and growth conditions
2.1.1 Plant materials
2.1.2 Plant growth conditions
2.2 Experimental methods
2.2.1 Cloning of the target fragment
2.2.1.1 Polymerase chain amplification (PCR)
2.2.1.2 Agarose gel electrophoresis and recovery of PCR products
2.2.2 Construction, preparation and transformation of vectors
2.2.2.1 Vector construction: Gateway Cloning and T4 connection
2.2.2.2 Small amount of plasmid extraction
2.2.2.3 Preparation and chemical transformation of DH5α competent cells
2.2.2.4 Preparation of Agrobacterium C58C1 competent cells and transformation byelectric shock
2.2.2.5 Agrobacterium-mediated transformation of Arabidopsis thaliana
2.2.3 Plant genomic DNA extraction
2.2.4 Identification of homozygotes for T-DNA insertion mutants
2.2.5 CRISPR-Cas9 system edits target gene
2.2.6 Extraction, electrophoretic detection and reverse transcription of plant RNA
2.2.6.1 Extraction of plant RNA
2.2.6.2 RNA electrophoresis
2.2.6.3 RNA reverse transcription
2.2.7 Real-time PCR
2.2.8 GUS staining experiment
2.2.9 Observation of GFP subcellular localization
2.2.10 Chromatin immunoprecipitation (ChIP)
2.2.10.1 Materials and reagents
2.2.11 Yeast-one-hybrid experiment (Y1H)
2.2.12 Plant N absorption and assimilation ability detection experiment
2.2.12.1 Metabolite analysis experiment
2.2.12.2 Chlorophyll content
2.2.12.3 Enzyme activity detection experiment
2.2.12.4 ~(15)N-nitrate or ~(156)N-ammonium uptake and ~(15)N accumulation
2.2.12.5 Chlorate sensitivity test
2.2.13 Field trial of rice
Chapter 3 Experimental Results
3.1 Functional analysis of OsNLPl
3.1.1 Verification of onlpl mutants and OsNLP1 overexpression lines
3.1.2 Expression pattern and subcellular localization of OsNLP1 in response to Navailability
3.1.3 Overexpression of the OsNLPl can complement the N starvation phenotype ofArabidopsis nlp7-1 mutant
3.1.4 OsNLP1 regulates plant growth in response to N availability
3.1.5 OsNLP1 is important for grain yield and NUE
3.1.6 OsNLP1 modulates the expression of N uptake and metabolism related genes
3.1.7 OsNLP1 directly binds to the nitrate response elements (NREs) in the promoter of Nuptake and assimilation related genes
3.1.8 OsNLPl genomic sequence has conserved SNPs between indica and japonica rice
3.2 Functional analysis of Arabidopsis NLP7 (AtNLP7) in cotton
3.2.1 Generation of the transgenic cotton plants that express AtNLP7
3.2.2 AtNLP7 enhances Cotton plant biomass under both low and high N conditions
3.2.3 Expression of AtNLP7 in cotton positively regulates genes involved in N signalingand assimilation
3.2.4 AtNLP7 improves cotton-growth in soil
3.2.5 AtNLP7 improves N and carbon assimilation of cotton plants
Chapter 4 Discussion
4.1 OsNLP1 is responsive to ammonium avialability and improves the NUE
4.2 OsNLPl positively regulates the transcription of genes related to nitrate uptake andassimilation
4.3 OsNLPl genomic sequence conserved SNPs between indica and japonica rice
4.4 The role of AtNLP7 in cotton plants
4.5 Summary
References
Appendix
Appendix 1 Abbreviations
Appendix 2 Related Medium Formulas
1. Plant growth medium formula
2. Bacteria growth medium
3. Yeast-two-hybrid culture medium formula
4. Formulation of plant DNA extraction buffer
5. GUS staining reagents
Appendix 3 Predicted NRE sequences in the promoter of NLP-regulated genes
Appendix 4 Primers used in experiments
Acknowledgements
Academic papers and other research results obtained during the period of study
【参考文献】：
期刊论文
[1]Timing and splitting of nitrogen fertilizer supply to increase crop yield and efficiency of nitrogen utilization in a wheat–peanut relay intercropping system in China[J]. Zhaoxin Liu,Fang Gao,Yan Liu,Jianqun Yang,Xiaoyu Zhen,Xinxin Li,Ying Li,Jihao Zhao,Jinrong Li,Bichang Qian,Dongqing Yang,Xiangdong Li. The Crop Journal. 2019(01)
[2]Molecular Basis and Regulation of Ammonium Transporter in Rice[J]. Mike MERRICK. Rice Science. 2009(04)
本文编号：3516277
【文章来源】：中国科学技术大学安徽省 211工程院校 985工程院校
【文章页数】：118 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Chapter 1 Introduction
1.1 The importance of nitrogen in plant growth and agricultural production
1.2 Plant N use efficiency
1.3 Plant N absorption and metabolic pathways
1.3.1 Nitrate nitrogen absorption and metabolism
1.3.2 Ammonium nitrogen absorption and metabolism
1.3.3 N and carbon metabolism balance
1.3.4 Research status of nitrate signaling in plants
1.4 Introduction of plant RWP-RK transcription factor family
1.4.1 Relationship and typical structural features of thresholds in RWP-RK family system
1.4.2 The Function of RWP-RK protein
1.5 The Function of NLPs in rice and cotton crops
Chapter 2 Experimental Materials and Methods
2.1 Plant materials and growth conditions
2.1.1 Plant materials
2.1.2 Plant growth conditions
2.2 Experimental methods
2.2.1 Cloning of the target fragment
2.2.1.1 Polymerase chain amplification (PCR)
2.2.1.2 Agarose gel electrophoresis and recovery of PCR products
2.2.2 Construction, preparation and transformation of vectors
2.2.2.1 Vector construction: Gateway Cloning and T4 connection
2.2.2.2 Small amount of plasmid extraction
2.2.2.3 Preparation and chemical transformation of DH5α competent cells
2.2.2.4 Preparation of Agrobacterium C58C1 competent cells and transformation byelectric shock
2.2.2.5 Agrobacterium-mediated transformation of Arabidopsis thaliana
2.2.3 Plant genomic DNA extraction
2.2.4 Identification of homozygotes for T-DNA insertion mutants
2.2.5 CRISPR-Cas9 system edits target gene
2.2.6 Extraction, electrophoretic detection and reverse transcription of plant RNA
2.2.6.1 Extraction of plant RNA
2.2.6.2 RNA electrophoresis
2.2.6.3 RNA reverse transcription
2.2.7 Real-time PCR
2.2.8 GUS staining experiment
2.2.9 Observation of GFP subcellular localization
2.2.10 Chromatin immunoprecipitation (ChIP)
2.2.10.1 Materials and reagents
2.2.11 Yeast-one-hybrid experiment (Y1H)
2.2.12 Plant N absorption and assimilation ability detection experiment
2.2.12.1 Metabolite analysis experiment
2.2.12.2 Chlorophyll content
2.2.12.3 Enzyme activity detection experiment
2.2.12.4 ~(15)N-nitrate or ~(156)N-ammonium uptake and ~(15)N accumulation
2.2.12.5 Chlorate sensitivity test
2.2.13 Field trial of rice
Chapter 3 Experimental Results
3.1 Functional analysis of OsNLPl
3.1.1 Verification of onlpl mutants and OsNLP1 overexpression lines
3.1.2 Expression pattern and subcellular localization of OsNLP1 in response to Navailability
3.1.3 Overexpression of the OsNLPl can complement the N starvation phenotype ofArabidopsis nlp7-1 mutant
3.1.4 OsNLP1 regulates plant growth in response to N availability
3.1.5 OsNLP1 is important for grain yield and NUE
3.1.6 OsNLP1 modulates the expression of N uptake and metabolism related genes
3.1.7 OsNLP1 directly binds to the nitrate response elements (NREs) in the promoter of Nuptake and assimilation related genes
3.1.8 OsNLPl genomic sequence has conserved SNPs between indica and japonica rice
3.2 Functional analysis of Arabidopsis NLP7 (AtNLP7) in cotton
3.2.1 Generation of the transgenic cotton plants that express AtNLP7
3.2.2 AtNLP7 enhances Cotton plant biomass under both low and high N conditions
3.2.3 Expression of AtNLP7 in cotton positively regulates genes involved in N signalingand assimilation
3.2.4 AtNLP7 improves cotton-growth in soil
3.2.5 AtNLP7 improves N and carbon assimilation of cotton plants
Chapter 4 Discussion
4.1 OsNLP1 is responsive to ammonium avialability and improves the NUE
4.2 OsNLPl positively regulates the transcription of genes related to nitrate uptake andassimilation
4.3 OsNLPl genomic sequence conserved SNPs between indica and japonica rice
4.4 The role of AtNLP7 in cotton plants
4.5 Summary
References
Appendix
Appendix 1 Abbreviations
Appendix 2 Related Medium Formulas
1. Plant growth medium formula
2. Bacteria growth medium
3. Yeast-two-hybrid culture medium formula
4. Formulation of plant DNA extraction buffer
5. GUS staining reagents
Appendix 3 Predicted NRE sequences in the promoter of NLP-regulated genes
Appendix 4 Primers used in experiments
Acknowledgements
Academic papers and other research results obtained during the period of study
【参考文献】：
期刊论文
[1]Timing and splitting of nitrogen fertilizer supply to increase crop yield and efficiency of nitrogen utilization in a wheat–peanut relay intercropping system in China[J]. Zhaoxin Liu,Fang Gao,Yan Liu,Jianqun Yang,Xiaoyu Zhen,Xinxin Li,Ying Li,Jihao Zhao,Jinrong Li,Bichang Qian,Dongqing Yang,Xiangdong Li. The Crop Journal. 2019(01)
[2]Molecular Basis and Regulation of Ammonium Transporter in Rice[J]. Mike MERRICK. Rice Science. 2009(04)
本文编号：3516277
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