枳碱性/中性转化酶基因PtrA/NINV功能分析

发布时间：2021-09-28 03:34

　　Poncirus trifoliate（L.）Raf在柑橘培养中广泛用作砧木,在充分冷驯化后可以抵御-20℃的低温。在前期工作中,本实验室利用抑制性消减杂交（SSH）的方法从枳中筛选出一种碱性/中性转化酶PtrA/NINV,但该基因的功能并不清楚。本研究从以下几个方面进行了研究:1、从枳中分离出PtrA/NINV的全长ORF序列和启动子区,鉴定并进行生物信息学分析。2、PtrA/NINV的亚细胞定位。3、PtrA/NINV的抗性分析(冷,盐,和干旱胁迫。主要结果如下:1.对PtrA/NINV进行序列分析,我们发现它编码一个等电点为6.59的并且具有678个氨基酸残基的蛋白质。这种蛋白质的分子量是76.4 kDa。PtrA/NINV以及其他来自不同高等植物和蓝藻的57个假定A/N-INV的蛋白研究表明PtrA/NINV被划分为一组,其成员存在于线粒体,并有氧化胁迫耐性。有调查了的的其它基因的PtrA/NINV以及其他基因的蛋白质序列的MEME分析多序列比对表明所有测试序列共享九个包含GH100保守结构的保守结构域（序1-9）。多重序列比对表明PtrA/NINV和其他测试序列之间有58-8... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：89 页

【学位级别】：硕士

【文章目录】：
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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