声明
英文缩略表
CHAPTER Ⅰ INTRODUCTION
1.1 Evolution and polyploid complexity Brassica napus
1.2 Regulation of fruit development in B. napus
1.3 Pod shattering mechanism
1.4 Genetic strategies to reduce pod shattering in B. napus
1.4.1 Genome editing tools
1.4.2 CRISPR system
1.4.3 Cas9
1.4.4 Application of CRISPR/Cas9 system in polyploid crops
1.5 Determining factors of pod shattering in B. napus
1.6 Assessment of shattering resistance index
1.6.1 Random impact test
1.7 Research basis
CHAPTER Ⅱ Functional Characterization of SHATTERPROOF Homoeologous for Pod Shatter Resistance in Brassica napus L. by Genome Editing
2.1 Introduction
2.2 Materials and Methods
2.2.1 Selection of target sequences and vector construction
2.2.2 Plant material, genetic transformation and plant growth
2.2.3 DNA extraction and mutant identification
2.2.4 Pod transverse section preparation
2.2.5 Random impact test
2.2.6 Statistical analysis
2.3. Results
2.3.1 Sequence analysis of BnSHP and vector construction
2.3.2 Mutation detection
2.3.3 Segregation pattern and inheritance of T1 mutations
2.3.4 BnSHP homoeologous display functional diversity for DZ development
2.3.5 Variation of pod shattering resistance in SHP mutants
2.3.6 Identification of transgene-free mutants
2.4. Discussion
2.5 Summary
CHAPTER Ⅲ Genome Editing of JAGGED Gene Reveals the Diverse Functional Role in Pod in B. napus L.
3.1 Introduction
3.2 Materials and Methods
3.2.1 sgRNA Design and Vector Construction
3.2.2 Plant material and vector transformation
3.2.3 DNA extraction and identification of positive mutant
3.2.4 Phenotypic characterization of pods
3.2.5 RNA isolation and quantitative real-time PCR
3.2.6 Staining of a transverse section of pods
3.2.7 Statistical analysis
3.3 Results
3.3.1 Sequence analysis of BnJAG gene
3.3.2 Knocking-out all five JAGGED homoeologs dramatically hampers pod
3.3.3 Mutagenesis in BnJAG.A08 enhances replum width in dehiscence zone
3.3.4 Inheritance pattern of mutagenesis at BnJAG.A08 homoeologs
3.3.5 Variation of pod phenotype and shattering resistance index (SRI)
3.3.6 Analysis of transgene-free mutants
3.4 Discussion
3.5 Summary
CHAPTER Ⅳ Genome Editing of INDEHISCENT Homoeologous Generates Transgene-free Shattering Resistant Phenotype in B. napus L.
4.1 Introduction
4.2 Materials and Methods
4.2.1 Phylogenetic analysis and selection of targets
4.2.2 Plant material and transformation
4.2.3 DNA extraction and identification of positive plants
4.2.4 RNA isolation and quantitative real-time PCR
4.2.5 Microscopy of pod developmental stages
4.2.6 Random impact test
4.2.7 Statistical t-test
4.3 Results
4.3.1 Sequence analysis of BnIND gene
4.3.2 Identification of CRISPR/Cas9 induced mutations
4.3.3 Expression pattern of pod shattering related genes
4.3.4 Determining of cell differentiation and lignified layer in dehiscence zone
4.3.5 Pod shattering resistance of mutants
4.3.6 Identification of transgene-free mutants
4.4 Discussion
4.5 Summary
CHAPTER Ⅴ Genome Editing of ALCATRAZ Homoeologous Partially Enhanced Shattering Resistance in B. napus L.
5.1 Introduction
5.2 Materials and Methods
5.2.1 Sequence identification and phylogenetic analysis
5.2.2 Plant material, vector construction and transformation
5.2.3 Mutant identification
5.2.4 Phenotypic observation and pod cross-section staining
5.2.5 Random impact test for pod shattering resistance
5.2.6 Statistical analysis
5.3. Results
5.3.1 Sequence analysis of BnALC, vector construction and transformation
5.3.2 Identification of CRISPR/Cas9 mutants
5.3.3 Valve margin development in pod
5.3.4 Pod shattering resistance of mutants
5.4 Discussion
5.5 Summary
CHAPTER Ⅵ Conclusion and Recommendations
6.1 Conclusion
6.2 Recommendations
参考文献
APPENDIX
致谢
CURRICULUM VITAE
PUBLICATION FROM DOCTORAL THESIS
中国农业科学院;
