二穗短柄草DREB基因家族分析和BdDREB1G在拟南芥中的过量表达

发布时间：2022-01-21 09:52

　　二穗短柄草属于禾本科,它与主要粮食作物如包括小麦、大麦和黑麦有密切的系统进化关系。二穗短柄草因其基因组小、生长周期短等优点而广泛用于研究。本研究在二穗短柄草DREB基因家族分析的基础上鉴定了58个BdDREB基因,根据进化关系可分为A1、A2、A3、A4、A5和A6六个亚组。染色体定位分析发现它们主要定位在二穗短柄草五个染色体上。对不同组织和不同非生物胁迫条件下表达模式分析发现他们主要参与植物的生长发育。此外,我们还成功克隆了BdDREB基因（XM₀03570049.3）,并成功在拟南芥中介导表达且筛选到一定数量的转基因拟南芥种子,最后用PCR检测和表型分析了转基因拟南芥植物,为进一步研究BdDERB的功能奠定了基础。 

【文章来源】：西北农林科技大学陕西省 211工程院校 985工程院校 教育部直属院校

【文章页数】：59 页

【学位级别】：硕士

【文章目录】：
ABSTRACT
摘要
Chapter one: Review of Literature
    1.1 Introduction
    1.2 Transcription factors (TFs)
        1.2.1 AP2/EREBP transcription factors
        1.2.2 AREB transcription factors
        1.2.3 NAC transcription factors
        1.2.4 WRKY transcription factors
        1.2.5 Zinc finger transcription factors
    1.3 Arabidopsis thaliana: the model plant
    1.4 Abiotic stresses
        1.4.1 Drought stress
        1.4.2 Salinity stress
        1.4.3 Cold stress
        1.4.4 Heat stress
    1.5 Research objectives and outline of the experimental design
        1.5.1 Research objectives
        1.5.2 Flow chart of research process
Chapter Two: Materials and Methods
    2.1 Materials
        2.1.1 Plant Materials and Growth Conditions
            2.1.1.1 Brachypodium distachyon Growth Conditions
            2.1.1.2 Arabidopsis thaliana Growth Conditions
            2.1.1.3 Sterilization Arabidopsis seeds
        2.1.2 Database software and online tools
        2.1.3 List of primers used in the present study
    2.2 Methods
        2.2.1 Bd DREB gene analysis
            2.2.1.1 Multiple sequence alignment and phylogenetic analysis
            2.2.1.2 Chromosome distribution analysis
        2.2.2 Overexpression Bd DREB1G gene in Arabidopsis
            2.2.2.1 General sterilization procedures
            2.2.2.2 Recombinant DNA techniques for cloning and DNA analysis
                2.2.2.2.1 RNA isolation and c DNA synthesis
                2.2.2.2.2 Quantitative real-time PCR analysis
            2.2.2.3 Gene cloning
            2.2.2.4 PCR product purification
            2.2.2.5 T/A cloning of PCR products
            2.2.2.6 Preparation of ultra-competent bacterial cells
            2.2.2.7 Transformation to E.coli
            2.2.2.8 Plasmid extraction
            2.2.2.9 Restriction enzymes digestion and ligation
                2.2.2.9.1 Plasmid extraction
                2.2.2.9.2 Agrobacterium
                2.2.2.9.3 Transformation to arabidopsis with MS media
                2.2.2.9.4 Transgenic plants
                    2.2.2.9.4.1 Arabidopsis transformation
                2.2.2.9.5 Screening of transgenic Arabidopsis and DNA extraction
        2.2.3 Statistical analysis
Chapter Three: Results and discussion
    3.1 Results
        3.1.1 the analysis of Bd DREB gene family
            3.1.1.1 Phylogenetic analysis
            3.1.1.2 Gene duplication and syntey analysis of Bd AP2 genes
        3.1.2 overexpression of Bd DREB1G in Arabidopsis thaliana
            3.1.2.1 Pre- experimental
            3.1.2.2 Cloning and sequence analysis of the Brachypodium DREB gene
            3.1.2.3 Screening and identification of transgenic Arabidopsis thaliana
                3.1.2.3.1 Screening test resistance to kanamycin
                3.1.2.3.2 PCR identified transgenic seedlings Arabidopsis
            3.1.2.4 Detection of gene expression in transgenic Arabidopsis thaliana
            3.1.2.5 Phenotypic analysis of transgenic Arabidopsis plants
    3.2 Discussion
Chapter Four: conclusions and recommendations
References
Acknowledgements
CURRICULUM VITAE



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