鸟氨酸氨基转移酶编码基因TaOAT的克隆及其与AtOAT基因在小麦中的功能分析
发布时间：2024-07-05 18:51
小麦是全球最重要的粮食作物之一,但小麦的产量却因环境胁迫的不断加重而倍受影响。因此,挖掘小麦抗逆基因资源对于提高小麦耐盐和抗旱能力非常必要。对生物胁迫和非生物胁迫的一个常见应对是脯氨酸(Pro)的积累。鸟氨酸氨基转移酶(OAT)又称为δ-鸟氨酸氨基转移酶(δOAT),是一种依赖于吡哆醛磷酸盐(PLP)的酶,参与鸟氨酸与谷氨酰5-半醛(GSA)的转换。鸟氨酸氨基转移酶是一种高度保守的酶,在脯氨酸(Pro)生物合成过程中催化鸟氨酸(Orn)转氨化为5-半醛(GSA)。该酶通过参与鸟氨酸途径,在逆境胁迫下植物产生的脯氨酸积累、细胞程序化死亡和非宿主疾病抗性中发挥作用。迄今为止,OAT基因已在水稻、玉米和高粱等植物中克隆并进行了功能鉴定,但在小麦中还没有关于OAT基因的研究。本研究拟对小麦中的OAT基因进行克隆和分子鉴定,并在小麦中分别对TaOAT和AtOAT基因进行功能分析。研究结果对于挖掘耐环境胁迫的基因资源和培育抗逆小麦新品种具有重要理论和实践意义。主要研究结果如下:1)通过生物信息分析、PCR扩增和测序验证等手段,在小麦第5部分同源群染色体上共鉴定了3个TaOAT同源基因,分别为TaO...
【文章页数】：124 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Abbreviations
Chapter 1:Introduction
1.1 Socioeconomic importance of wheat
1.2 Current status of wheat production and yield losses due to abiotic stresses
1.3 Strategies to combat the environmental stress in wheat
1.3.1 Transgenic technique as a potential tool to develop stress resistance cultivars
1.3.2 Limitation of transgenic technique in developing resistant wheat cultivars to abiotic stress conditions
1.3.3 Overcoming the limitation in wheat improvement by transgenic approach
1.4 Plant responses toward abiotic stresses
1.4.1 Proline metabolic adaptation in plants during stress
1.4.2 Proline biosynthesis pathway
1.5 Biological roles of OAT in plant stress tolerance
1.5.1 General kinetic property of OAT enzyme
1.5.2 OAT is highly conserved among prokaryotes and eukaryotes
1.5.3 OAT is linked with multiple metabolic pathways
1.6 Biological functions associated with OAT
1.6.1 OAT being involved in stress-induced proline accumulation
1.6.2 OAT being involved in plant non-host disease resistance
1.7 Aims and objectives of this study
Chapter 2 Cloning and molecular characterization of wheat ornithine amino transferase(TaOAT) encoded genes
2.1 Introduction
2.2 Materials and methods
2.2.1 Plant materials and vectors
2.2.2 Sequence retrieval
2.2.3 Extraction of gDNA
2.2.4 Extraction of total RNA and synthesis of cDNA
2.2.5 Cloning of full-length TaOAT genes
2.2.6 Chromosome localization
2.2.7 Subcellular localization of TaOAT
2.2.8 Phylogenetic analysis
2.2.9 Promoter analysis
2.2.10 Protein-protein interaction
2.2.11 Stress treatment and samples collection
2.2.12 Quantitative real-time RT-PCR(qRT-PCR)
2.3 Results
2.3.1 Isolation TaOAT genes in hexaploid wheat
2.3.2 Molecular structure of TaOAT genes
2.3.3 Chromosomal localization of TaOAT genes
2.3.4 In-silico analysis of subcellular localization of OAT genes in different species
2.3.5 Subcellular localization TaOAT gene
2.3.6 Phylogenetic analysis of wheat OAT genes
2.3.7 Promoter analysis of TaOAT genes
2.3.8 Protein-protein interactions of wheat OAT genes
2.3.9 Expression profile of TaOAT genes in different tissues at developmental stages
2.3.10 Expression patterns of TaOAT genes induced by exogenous PEG and Na Cl
2.4 Discussion
2.5 Summary
Chapter 3:Functional characterization of wheat ornithine amino transferase(TaOAT)encoded genes
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant materials and vectors
3.2.2 Construction of expression vector
3.2.3 Construction of CRISPR-Cas9 vector
3.2.4 Triparental mating
3.2.5 Agrobacterium-mediated wheat transformation
3.2.6 Detection of transgenic wheat plants
3.2.7 Screening of independent stable transgenic lines
3.2.8 Salt tolerant assay
3.2.9 Drought tolerance assay
3.3 Results
3.3.1 Generation of transgenic wheat plants
3.3.2 Generation of stable transgenic lines containing pWMB206 vector
3.3.3 Generation of stable transgenic lines containing pWMB220 vector
3.3.4 Transgenic plants containing pWMB206 showed enhanced tolerance to salt stress in vitro condition
3.3.5 Transgenic plants showed an enhanced tolerance to drought
3.4 Discussion
3.5 Summary
Chapter 4:Expression and functional analysis of Arabidopsis ornithine aminotransferase(AtOAT)encoded gene in wheat
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant materials
4.2.2 Plasmid construction
4.2.3 Triparental mating
4.2.4 Agrobacterium-mediated transformation of wheat immature embryos
4.2.5 Detection of transgenic plants by Quick Stix strip
4.2.6 Identification of transgenic plants by PCR amplification
4.2.7 Generation of marker free T1 transgenic plants
4.2.8 Generation of homozygous transgenic plants by chromosome elimination
4.2.9 Chromosome preparation and fluorescence in situ hybridization
4.2.10 Semi-quantitative PCR analysis of AtOAT in the stable transgenic lines
4.2.11 Drought treatment assay
4.2.12 Salt treatment assay
4.2.13 Heat treatment assay
4.2.14 RNA extraction and expression profile of marker genes under different stress
4.2.15 Statistical analysis
4.3 Results
4.3.1 Identification of expression vector
4.4 Discussion
4.5 Summary
Conclusions
References
Acknowledgement
Resume
本文编号：4001373
【文章页数】：124 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Abbreviations
Chapter 1:Introduction
1.1 Socioeconomic importance of wheat
1.2 Current status of wheat production and yield losses due to abiotic stresses
1.3 Strategies to combat the environmental stress in wheat
1.3.1 Transgenic technique as a potential tool to develop stress resistance cultivars
1.3.2 Limitation of transgenic technique in developing resistant wheat cultivars to abiotic stress conditions
1.3.3 Overcoming the limitation in wheat improvement by transgenic approach
1.4 Plant responses toward abiotic stresses
1.4.1 Proline metabolic adaptation in plants during stress
1.4.2 Proline biosynthesis pathway
1.5 Biological roles of OAT in plant stress tolerance
1.5.1 General kinetic property of OAT enzyme
1.5.2 OAT is highly conserved among prokaryotes and eukaryotes
1.5.3 OAT is linked with multiple metabolic pathways
1.6 Biological functions associated with OAT
1.6.1 OAT being involved in stress-induced proline accumulation
1.6.2 OAT being involved in plant non-host disease resistance
1.7 Aims and objectives of this study
Chapter 2 Cloning and molecular characterization of wheat ornithine amino transferase(TaOAT) encoded genes
2.1 Introduction
2.2 Materials and methods
2.2.1 Plant materials and vectors
2.2.2 Sequence retrieval
2.2.3 Extraction of gDNA
2.2.4 Extraction of total RNA and synthesis of cDNA
2.2.5 Cloning of full-length TaOAT genes
2.2.6 Chromosome localization
2.2.7 Subcellular localization of TaOAT
2.2.8 Phylogenetic analysis
2.2.9 Promoter analysis
2.2.10 Protein-protein interaction
2.2.11 Stress treatment and samples collection
2.2.12 Quantitative real-time RT-PCR(qRT-PCR)
2.3 Results
2.3.1 Isolation TaOAT genes in hexaploid wheat
2.3.2 Molecular structure of TaOAT genes
2.3.3 Chromosomal localization of TaOAT genes
2.3.4 In-silico analysis of subcellular localization of OAT genes in different species
2.3.5 Subcellular localization TaOAT gene
2.3.6 Phylogenetic analysis of wheat OAT genes
2.3.7 Promoter analysis of TaOAT genes
2.3.8 Protein-protein interactions of wheat OAT genes
2.3.9 Expression profile of TaOAT genes in different tissues at developmental stages
2.3.10 Expression patterns of TaOAT genes induced by exogenous PEG and Na Cl
2.4 Discussion
2.5 Summary
Chapter 3:Functional characterization of wheat ornithine amino transferase(TaOAT)encoded genes
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant materials and vectors
3.2.2 Construction of expression vector
3.2.3 Construction of CRISPR-Cas9 vector
3.2.4 Triparental mating
3.2.5 Agrobacterium-mediated wheat transformation
3.2.6 Detection of transgenic wheat plants
3.2.7 Screening of independent stable transgenic lines
3.2.8 Salt tolerant assay
3.2.9 Drought tolerance assay
3.3 Results
3.3.1 Generation of transgenic wheat plants
3.3.2 Generation of stable transgenic lines containing pWMB206 vector
3.3.3 Generation of stable transgenic lines containing pWMB220 vector
3.3.4 Transgenic plants containing pWMB206 showed enhanced tolerance to salt stress in vitro condition
3.3.5 Transgenic plants showed an enhanced tolerance to drought
3.4 Discussion
3.5 Summary
Chapter 4:Expression and functional analysis of Arabidopsis ornithine aminotransferase(AtOAT)encoded gene in wheat
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant materials
4.2.2 Plasmid construction
4.2.3 Triparental mating
4.2.4 Agrobacterium-mediated transformation of wheat immature embryos
4.2.5 Detection of transgenic plants by Quick Stix strip
4.2.6 Identification of transgenic plants by PCR amplification
4.2.7 Generation of marker free T1 transgenic plants
4.2.8 Generation of homozygous transgenic plants by chromosome elimination
4.2.9 Chromosome preparation and fluorescence in situ hybridization
4.2.10 Semi-quantitative PCR analysis of AtOAT in the stable transgenic lines
4.2.11 Drought treatment assay
4.2.12 Salt treatment assay
4.2.13 Heat treatment assay
4.2.14 RNA extraction and expression profile of marker genes under different stress
4.2.15 Statistical analysis
4.3 Results
4.3.1 Identification of expression vector
4.4 Discussion
4.5 Summary
Conclusions
References
Acknowledgement
Resume
本文编号：4001373
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