氮（N）是植物正常生長發(fā)育和農(nóng)業(yè)生產(chǎn)所必需的一種大量元素。農(nóng)民在農(nóng)業(yè)生產(chǎn)中施用大量的氮肥以維持作物的高產(chǎn)。但是,由于氮肥利用效率過低,大部分沒有被植物吸收的氮肥流失到環(huán)境中,造成嚴重的環(huán)境污染并且大大增加了農(nóng)業(yè)生產(chǎn)的成本。解決這些問題的一個理想且經(jīng)濟的解決方案是提高作物的氮利用效率,這一直是全世界科學家研究的焦點,但關于它的遺傳決定因素和調(diào)控機理還有待進一步的探究。水稻是世界上最重要的糧食作物之一,為30億人提供35%-60%的熱量。提高水稻的氮利用效率是很有必要的。我們的工作表明了水稻NIN-LIKE PROTEIN 1（OsNLP1）在提高NUE過程中起著核心的作用。OsNLP1蛋白定位于細胞核,N饑餓處理可以在轉錄水平迅速的誘導其表達。在不同施氮條件下,過量表達OsNLP1都提高了水稻的生長、籽粒產(chǎn)量和NUE,而敲除OsNLP1則降低了低氮條件下的籽粒產(chǎn)量和NUE。OsNLP1通過協(xié)同調(diào)控多個氮素吸收和同化的基因來調(diào)控硝態(tài)氮和銨態(tài)氮的利用。染色質免疫沉淀和酵母單雜交實驗分析表明,OsNLP1可以直接結合這些基因的啟動子來激活它們的表達。因此,我們的實驗結果表明OsNLP1是氮素利... 

【文章來源】：中國科學技術大學安徽省 211工程院校 985工程院校

【文章頁數(shù)】：118 頁

【學位級別】：博士

【文章目錄】：
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


【參考文獻】：
期刊論文
[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)



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