声明
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
2.3.1 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 NaCl
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 QuickStix 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
参考文献
致谢
Resume
中国农业科学院;
