OsbZIP46CA1和SAPK6共超量表达水稻的非生物逆境抗性评价

发布时间：2024-02-15 10:05

　　水稻(Oryza sativa L.)是世界上最重要的粮食作物之一,为世界一半以上的人口提供主要食物。然而,全球气候的不断变化给水稻生产带来重大挑战,特别是干旱环境,这严重影响了世界经济和粮食安全。对水稻的抗逆性状进行遗传改良是应对这一状况的主要策略之一。根据已有报道,水稻抗旱性状是由多基因控制的,因此,多基因聚合的策略在理论上可以用于改善水稻的抗旱性。然而,目前这种策略仍然缺乏实验证据。在本研究中,我们通过MISSA(multiple-round in vivo sitespecific assembly)系统组装了从水稻中分离的几个干旱应答基因,并且通过农杆菌介导转化法将聚合载体以及对应的单基因超表达载体导入KY131水稻品种中。之后,我们鉴定了T1代转基因植株中的目的基因转录水平和插入片段拷贝数,并选取单拷贝、超量表达目的基因的转基因家系和阴性转基因材料(KY131-N)用于大田干旱胁迫的预实验和之后的进一步实验中。在预实验期间,通过对叶片死亡程度和结实率的目测,我们发现共表达两个基因OsbZIP46CA1(编码bZIP转录因子OsbZIP46的组成型激活形式)和SAPK6(编码...

【文章页数】：111 页

【学位级别】：博士

【文章目录】：
Abstract in English
Abstract in Chinese
List of Abbreviations
1 Introduction
    1.1 The origin of the research issue
    1.2 Literature review
        1.2.1 Responses of plant under abiotic stresses
            1.2.1.1 Responses and signal transduction of plants under abiotic stresses
            1.2.1.2 Regulatory network of gene expression under abiotic stresses
        1.2.2 The role of ABA on abiotic stress response in plant
        1.2.3 Genetic transformation in plants
        1.2.4 Progress of technology study for the introduction of multiple genes into transgenic plants
        1.2.5 Candidate genes for genetic transformation
            1.2.5.1 bZIP transcription factor-encoding genes
            1.2.5.2 SAPK protein kinase-encoding genes
    1.3 Research purpose and significance
2 Materials and methods
    2.1 Experimental materials
    2.2 Agrobacterium-mediated transformation
    2.3 DNA extraction and PCR positive test
    2.4 RNA extraction, reverse transcription and Real-time PCR
    2.5 Southern blot hybridization
    2.6 ABA sensitivity test of the transgenic plants
    2.7 Stress tolerance experiments of transgenic rice at the seedling stage
        2.7.1 Drought resistance experiment of transgenic rice at the seedling stage
        2.7.2 Temperature stress resistance experiments of transgenic rice at the seedling stage
    2.8 Drought resistance experiment of transgenic rice at the reproductive stage
    2.9 Determination of stress-related physiological indicators in rice
        2.9.1 Determination of water loss rate in rice leaves
        2.9.2 Determination of cell membrane permeability
        2.9.3 Measure content of malondialdehyde (MDA) in rice leaves
    2.10 RNA-Seq and analysis
3 Results
    3.1 Screening of candidate genes and stress resistance in the transgenic plants
        3.1.1 Identification the presence of candidate genes in the transgenic plants
        3.1.2 Expression level analysis of candidate genes in the transgenic plants
        3.1.3 Copy number analysis of the partial T1 transgenic plants
        3.1.4 Phenotypic pre-evaluation of transgenic materials in the field
    3.2 Increased ABA sensitivity of XL22 co-overexpression seedlings
    3.3 Enhanced drought resistance of the XL22 transgenic seedlings
    3.4 Enhanced drought resistance of the XL22 transgenic plants at the reproductive stage
    3.5 The XL22 transgenic lines improved heat and cold tolerance
    3.6 Enhanced oxidation resistance of XL22 plants under heat stress
    3.7 Transcriptome profiling of the XL22 transgenic plants
4 Discussion
    4.1 Co-overexpression of OsbZIP46CA1 and SAPK6 enhances drought tolerance
    4.2 Rice drought resistance evaluation indicators
    4.3 Co-overexpression of OsbZIP46CA1 and SAPK6 improves tolerance to temperature stress
    4.4 Application of multi-gene assembly strategy in improving stress resistance of rice
References
Appendices
    Appendix 1. Supplemental figures
    Appendix 2. Supplemental datas
    Appendix 3. Protocols
        Protocol 1. Plasmid DNA extraction
        Protocol 2. Extraction of total DNA using Plant DNAzol reagent
        Protocol 3. Southern blot hybridization
Resume
Acknowledgement



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