小麦ABA受体基因TaPYL3的克隆和功能分析
发布时间：2022-02-24 23:33
普通小麦（Triticum aestivum L.）是全球重要的栽培作物之一,为人类提供了主要的能源物质。中国是世界上最大的小麦生产和消费国家。小麦生产中生物和非生物胁迫导致小麦产量低于潜在产量,干旱胁迫是其中一个主要原因。从小麦种质资源中发掘抗旱基因并解析其功能是提高小麦抗旱性的有效途径。PYL（pyrabactin resistance（PYR）like）/PYR（pyrabactin resistance）/RCAR（regulatory component of ABA receptor）属于ABA受体类蛋白,拟南芥中该蛋白家族共有14个成员,在干旱胁迫条件下参与激活ABA信号途径。本研究参考AtPYL3基因序列,在小麦中克隆到位于A基因组上的同源基因,命名为TaPYL3。通过分析小麦幼苗的基因表达模式,发现TaPYL3能够响应逆境胁迫。利用过表达TaPYL3基因的拟南芥T3代株系调查胁迫处理的幼苗表型。对小麦群体TaPYL3基因测序,利用其多态性位点开发分子标记,分析了分子标记与农艺性状的相关性。主要研究结果如下:1、TaPYL3基因序列全长3115 b...
【文章来源】：中国农业科学院北京市
【文章页数】：93 页
【学位级别】：博士
【文章目录】：
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
本文编号：3643671
【文章来源】：中国农业科学院北京市
【文章页数】：93 页
【学位级别】：博士
【文章目录】：
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
本文编号：3643671
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