Poncirus trifoliate（L.）Raf在柑橘培養(yǎng)中廣泛用作砧木,在充分冷馴化后可以抵御-20℃的低溫。在前期工作中,本實(shí)驗(yàn)室利用抑制性消減雜交（SSH）的方法從枳中篩選出一種堿性/中性轉(zhuǎn)化酶PtrA/NINV,但該基因的功能并不清楚。本研究從以下幾個方面進(jìn)行了研究:1、從枳中分離出PtrA/NINV的全長ORF序列和啟動子區(qū),鑒定并進(jìn)行生物信息學(xué)分析。2、PtrA/NINV的亞細(xì)胞定位。3、PtrA/NINV的抗性分析(冷,鹽,和干旱脅迫。主要結(jié)果如下:1.對PtrA/NINV進(jìn)行序列分析,我們發(fā)現(xiàn)它編碼一個等電點(diǎn)為6.59的并且具有678個氨基酸殘基的蛋白質(zhì)。這種蛋白質(zhì)的分子量是76.4 kDa。PtrA/NINV以及其他來自不同高等植物和藍(lán)藻的57個假定A/N-INV的蛋白研究表明PtrA/NINV被劃分為一組,其成員存在于線粒體,并有氧化脅迫耐性。有調(diào)查了的的其它基因的PtrA/NINV以及其他基因的蛋白質(zhì)序列的MEME分析多序列比對表明所有測試序列共享九個包含GH100保守結(jié)構(gòu)的保守結(jié)構(gòu)域（序1-9）。多重序列比對表明PtrA/NINV和其他測試序列之間有58-8... 

【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁數(shù)】：89 頁

【學(xué)位級別】：碩士

【文章目錄】：
摘要
Abstract
Abbreviations
1. Introduction
2. Literature review
    2.1. The common approach of functional characterization of the gene
    2.2. Abiotic stress: As major environmental factors
        2.2.1. Drought stress
        2.2.2. Salt stress
        2.2.3. Cold stress
    2.3. Plant responses to unfavorable abiotic stressors
        2.3.1. Generic pathway of plant response to abiotic stresses
        2.3.2. Osmotic response of plants against abiotic stress
        2.3.3. Reactive oxygen species (ROS) homoeostasis under abiotic stress
    2.4. Photosynthesis process and its related parameters
    2.5. Alkaline/neutral invertase genes
3. The objectives
4. Materials and methods
    4.1. Materials
        4.1.1. Plant materials and growth conditions
        4.1.2. Plasmids
        4.1.3. Bacteria
        4.1.4. Kits and Enzymes
    4.2. Methods
        4.2.1. Bioinformatics analysis of Ptr A/NINV sequence
        4.2.2. Transcription level analysis of Ptr A/NINV
            4.2.2.1. RNA extraction
            4.2.2.2. First strand c DNA synthesis
            4.2.2.3. Quality test of synthesized c DNA
            4.2.2.4. Quantitative real-time RT-PCR (q RT-PCR) analysis
        4.2.3. Subcellular localization analysis of Ptr A/NINV
            4.2.3.1. Construct the fusion protein of Ptr A/NINV:: GFP
            4.2.3.2. Agrobacterium-mediated transient expression of the Ptr A/NINV:: GFP fusion protein
        4.2.4. Identification the promoter sequence of Ptr A/NINV
            4.2.4.1. DNA extraction
            4.2.4.2. Isolation the promoter region of Ptr A/NINV
        4.2.5. Isolation of Ptr A/NINV protein
            4.2.5.1. Construct the recombinant His6:: Ptr A/NINV fusion protein
            4.2.5.2. Assay the recombinant His6:: Ptr A/NINV fusion protein
        4.2.6. Abiotic stress tolerance analysis of Ptr A/NINV overexpressing plants
            4.2.6.1. Regeneration of Ptr A/NINV overexpressing plants
            4.2.6.2. Cold stress tolerance assay
            4.2.6.3. Salt stress tolerance assay
            4.2.6.4. Drought stress tolerance assay
            4.2.6.5. Physiological and biochemical measurements
        4.2.7. Statistical analysis
5. Results
    5.1. Identification and sequence analysis of Ptr A/NINV
    5.2. Identification the promoter sequence of Ptr A/NINV
    5.3. Expression pattern analysis of Ptr A/NINV
    5.4. Ptr A/NINV localizes in the mitochondria and chloroplast
    5.5. Generation of stable Ptr A/NINV-overexpressing plants
    5.6. Enhanced cold tolerance in the transgenic plants
    5.7. Ptr A/NINV overexpression renders robust salt stress tolerance
    5.8. Overexpression of Ptr A/NINV improved drought tolerance
    5.9. Ptr A/NINV-overexpressing plants accumulate lower ROS and hold moreantioxidant enzyme activities
    5.10. The increase in A/N-INV activity and reducing sugar contents underpin thedetoxification of ROS
    5.11. Analysis of the expression patterns of several genes related to antioxidantdefense, stress response and sugar metabolism under cold and salt stresses
6. Discussion
7. Conclusion
8. References
9. The achievements
10. Appendixes
    Appendix-I
    Appendix-II
    Appendix-III
    Appendix-IV
    Appendix-V
    Appendix-VI
    Appendix-VII
    Appendix-VIII
ACKNOWLEDGMENTS



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