普通小麥（Triticum aestivum L.）是全球重要的栽培作物之一,為人類提供了主要的能源物質(zhì)。中國是世界上最大的小麥生產(chǎn)和消費國家。小麥生產(chǎn)中生物和非生物脅迫導(dǎo)致小麥產(chǎn)量低于潛在產(chǎn)量,干旱脅迫是其中一個主要原因。從小麥種質(zhì)資源中發(fā)掘抗旱基因并解析其功能是提高小麥抗旱性的有效途徑。PYL（pyrabactin resistance（PYR）like）/PYR（pyrabactin resistance）/RCAR（regulatory component of ABA receptor）屬于ABA受體類蛋白,擬南芥中該蛋白家族共有14個成員,在干旱脅迫條件下參與激活A(yù)BA信號途徑。本研究參考AtPYL3基因序列,在小麥中克隆到位于A基因組上的同源基因,命名為TaPYL3。通過分析小麥幼苗的基因表達(dá)模式,發(fā)現(xiàn)TaPYL3能夠響應(yīng)逆境脅迫。利用過表達(dá)TaPYL3基因的擬南芥T₃代株系調(diào)查脅迫處理的幼苗表型。對小麥群體TaPYL3基因測序,利用其多態(tài)性位點開發(fā)分子標(biāo)記,分析了分子標(biāo)記與農(nóng)藝性狀的相關(guān)性。主要研究結(jié)果如下:1、TaPYL3基因序列全長3115 b... 

【文章來源】：中國農(nóng)業(yè)科學(xué)院北京市

【文章頁數(shù)】：93 頁

【學(xué)位級別】：博士

【文章目錄】：
摘要
abstract
LIST OF ABBREVIATION
Chapter 1 Introduction
    1.1 Wheat domestication and evolution
    1.2 Wheat importance and production
    1.3 Impact of abiotic stresses in crops
    1.4 Wheat plants strategies to tackle drought stress
    1.5 ABA hormone and its role under abiotic stress
    1.6 ABA receptors in plants
    1.7 Structure and function of PYL
    1.8 Role of PYL in ABA signaling pathway
    1.9 Role of PYL in gene expression and transcriptional responses under abiotic stress
    1.10 Significance of research work
    1.11 Research objective
Chapter 2 TaPYL3 expression and bioinformatics analyses
    2.1 Introduction to Quantitative Real-Time PCR
        2.1.1 Gene regulation pattern in PYL receptors
        2.1.2 Bioinformatics analysis in gene families
        2.1.3 Research objectives
    2.2 Materials and methods
        2.2.1 Plant materials
        2.2.2 Treatment and conditions
        2.2.3 RNA extraction
        2.2.4 Quantification of RNA on nano-drop spectrophotometer
        2.2.5 c DNA synthesis
        2.2.6 Quantitative real-time PCR (q RT- PCR)
        2.2.7 Primers designed for gene expression analyses by q RT-PCR
        2.2.8 Sequence analysis and alignment of Ta PYL3
        2.2.9 Phylogenetic tree construction
    2.3 Results
        2.3.1 Ta PYL3 sequence alignment
        2.3.2 Phylogenetic analysis
        2.3.3 Ta PYL3 response to PEG treatment
        2.3.4 Ta PYL3 response to Na Cl treatment
        2.3.5 Ta PYL3 response to cold treatment
        2.3.6 Ta PYL3 response to heat stress
        2.3.7 Ta PYL3 response to ABA treatment
    2.4 Discussion
    2.5 Conclusion
Chapter 3 Abiotic stress tolerance in transgenic Arabidopsis
    3.1 Introduction
        3.1.1 Phenotypes of transgenic plants under abiotic stress
    3.2 Materials and Methods
        3.2.1 Transgenic plant material cultivation
        3.2.2 Isolation of Ta PYL3 candidate sequence
        3.2.3 Primers used
        3.2.4 Extraction of wheat genomic DNA
        3.2.5 PCR and agarose gel electrophoresis
        3.2.6 Purification of PCR product
        3.2.7 Cloning and heat shock transformation
        3.2.8 Heat shock transformation protocol
        3.2.9 Colony PCR to confirm the positive plasmid with gene insert
        3.2.10 Plasmid extraction
        3.2.11 Plasmid DNA sequencing
        3.2.12 PCR for generating plasmid vector construct
        3.2.13 Preparation of competent cells of A. tumefaciens
        3.2.14 Agrobacterium mediated transformation in Arabidopsis by floral dip method
        3.2.15 Screening for selection of transformed Arabidopsis seeds
        3.2.16 Seed sterilization
        3.2.17 Preparation of MS nutrient medium
        3.2.18 Tissue culture
        3.2.19 Abiotic stress treatment of Ta PYL3 transformed Arabidopsis plants
        3.2.20 Gene amplification and transcriptional expression in transgenic lines
        3.2.21 Relative expression level of transgenic Arabidopsis lines
    3.3 Results (Transgenic Arabidopsis)
        3.3.1 Mannitol stress treatment
        3.3.2 4°C stress treatment
        3.3.3 ABA stress treatment
        3.3.4 Na Cl stress treatment
        3.3.5 Gene amplification and relative expression in transgenic lines
    3.4 Discussion
    3.5 Conclusion
Chapter 4 Functional marker development of Ta PYL3 gene from wheat
    4.1 Introduction
        4.1.1 Development and application of molecular marker
        4.1.2 Natural variation in wheat by comparative genomics approach
    4.2 Materials and methods
        4.2.1 Plant materials
        4.2.2 Agronomic traits measurements of plant materials
        4.2.3 Primer specificity analysis
        4.2.4 Cloning and sequence analysis of Ta PYL3
        4.2.5 Development of a functional marker for Ta PYL3
        4.2.6 Gene linkage chromosome mapping
        4.2.7 Association analysis between Ta PYL3 allelic variation and agronomic traits
    4.3 Results
        4.3.1 Ta PYL3 genetic mapping
        4.3.2 Cloning and SNP identification of Ta PYL3 gene
        4.3.3 Functional marker development of Ta PYL3
        4.3.4 Association of SNP (G/A) at 1605 position with chlorophyll content and spikelet number per spike
        4.3.5 Association of SNP ( C/A) at 2547 position with grains per spike, thousand kernel weight and yield per plant
        4.3.6 Geographical sharing of G/A SNP allelic variation in Chinese wheat producing regions
        4.3.7 Geographical sharing of C/A SNP allelic variation in Chinese wheat producing regions
    4.4 Discussions
        4.4.1 Role of Ta PYL3 in plant species
        4.4.2 TaPYL3 cloning and functional marker development
        4.4.3 TaPYL3 is a drought tolerance gene and may improve wheat yield and quality
        4.4.4 Selection of TaPYL3 alleles in wheat agro-ecological zones
Chapter 5 Conclusion
REFERENCES
ACKNOWLEDGEMENT
RESUME



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