鳥氨酸氨基轉(zhuǎn)移酶編碼基因TaOAT的克隆及其與AtOAT基因在小麥中的功能分析
發(fā)布時間：2024-07-05 18:51
小麥?zhǔn)侨蜃钪匾募Z食作物之一,但小麥的產(chǎn)量卻因環(huán)境脅迫的不斷加重而倍受影響。因此,挖掘小麥抗逆基因資源對于提高小麥耐鹽和抗旱能力非常必要。對生物脅迫和非生物脅迫的一個常見應(yīng)對是脯氨酸(Pro)的積累。鳥氨酸氨基轉(zhuǎn)移酶(OAT)又稱為δ-鳥氨酸氨基轉(zhuǎn)移酶(δOAT),是一種依賴于吡哆醛磷酸鹽(PLP)的酶,參與鳥氨酸與谷氨酰5-半醛(GSA)的轉(zhuǎn)換。鳥氨酸氨基轉(zhuǎn)移酶是一種高度保守的酶,在脯氨酸(Pro)生物合成過程中催化鳥氨酸(Orn)轉(zhuǎn)氨化為5-半醛(GSA)。該酶通過參與鳥氨酸途徑,在逆境脅迫下植物產(chǎn)生的脯氨酸積累、細(xì)胞程序化死亡和非宿主疾病抗性中發(fā)揮作用。迄今為止,OAT基因已在水稻、玉米和高粱等植物中克隆并進行了功能鑒定,但在小麥中還沒有關(guān)于OAT基因的研究。本研究擬對小麥中的OAT基因進行克隆和分子鑒定,并在小麥中分別對TaOAT和AtOAT基因進行功能分析。研究結(jié)果對于挖掘耐環(huán)境脅迫的基因資源和培育抗逆小麥新品種具有重要理論和實踐意義。主要研究結(jié)果如下:1)通過生物信息分析、PCR擴增和測序驗證等手段,在小麥第5部分同源群染色體上共鑒定了3個TaOAT同源基因,分別為TaO...
【文章頁數(shù)】：124 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：4001373
【文章頁數(shù)】：124 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：4001373
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