CRISPR/Cas9基因编辑系统提高油菜的抗裂角性
发布时间：2022-01-14 00:25
油菜角果在收获时段易裂会造成严重的产量损失。油菜角果易裂与角果离区细胞降解密切相关。一些基因,诸如Bn JAG,Bn SHP,Bn IND和Bn ALC都参与到角果离区形成及发育中。为了提高油菜抗裂角性,利用CRISPR/Cas9系统将Bn JAG,Bn SHP,Bn IND和Bn ALC分别进行敲除。对油菜中Bn JAG所有拷贝进行敲除后,角果的大小及侧生组织发育受到很大影响。整个角果成“愈伤”状,没有明显的果瓣,隔膜和离区。此外,角果隔膜不能延伸到整个角果,隔膜两侧产生假种子,这些种子不能发育成熟。然而,当只有Bn JAG.A08-NUB-Like（Bn JAG-A08）突变时,隔膜发育完好,延伸到整个角果,将角果分成两半。整个角果相对于野生型变短变粗。为了探讨Bn SHP基因功能,我们将Bn SHP1-157（Bn SHP1-A09突变）和Bn SHP4-484（四个Bn SHP基因突变系）杂交,产生五个Bn SHP突变系（Bn SHP5-184）。Bn SHP5-184中,离区木质化层和离区分离层退化,角果SRI值为0.31显著高于野生型（SRI=0.036）。此外,对Bn S...
【文章来源】：中国农业科学院北京市
【文章页数】：115 页
【学位级别】：博士
【文章目录】：
DEDICATION
摘要
ABSTRACT
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
1.4.3Cas9
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 Bn SHP and vector construction
2.3.2 Mutation detection
2.3.3 Segregation pattern and inheritance of T1 mutations
2.3.4 Bn SHP 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 sg RNA 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 Bn JAG gene
3.3.2 Knocking-out five Bn JAG homoeologs hampers pod development
3.3.3 Mutagenesis in Bn JAG.A08 enhances replum width in dehiscence zone
3.3.4 Inheritance pattern of mutagenesis at Bn JAG.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 Bn IND 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 Bn ALC,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
REFERENCES
APPENDIX
ACKNOWLEDGEMENTS
CURRICULUM VITAE
PUBLICATION FROM DOCTORAL THESIS
【参考文献】：
期刊论文
[1]Genome editing opens a new era of genetic improvement in polyploid crops[J]. Qamar U.Zaman,Chao Li,Hongtao Cheng,Qiong Hu. The Crop Journal. 2019(02)
[2]Rapeseed research and production in China[J]. Qiong Hu,Wei Hua,Yan Yin,Xuekun Zhang,Lijiang Liu,Jiaqin Shi,Yongguo Zhao,Lu Qin,Chang Chen,Hanzhong Wang. The Crop Journal. 2017(02)
[3]油菜抗裂角性鉴定方法的改进及试验[J]. 彭鹏飞,李云昌,梅德圣,刘道敏,付丽,王会,桑世飞,陈玉峰,胡琼. 农业工程学报. 2013(21)
[4]甘蓝型油菜抗裂角品种(系)的筛选与分析[J]. 文雁成,傅廷栋,涂金星,马朝芝,沈竞雄,张书芬. 作物学报. 2008(01)
本文编号：3587428
【文章来源】：中国农业科学院北京市
【文章页数】：115 页
【学位级别】：博士
【文章目录】：
DEDICATION
摘要
ABSTRACT
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
1.4.3Cas9
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 Bn SHP and vector construction
2.3.2 Mutation detection
2.3.3 Segregation pattern and inheritance of T1 mutations
2.3.4 Bn SHP 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 sg RNA 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 Bn JAG gene
3.3.2 Knocking-out five Bn JAG homoeologs hampers pod development
3.3.3 Mutagenesis in Bn JAG.A08 enhances replum width in dehiscence zone
3.3.4 Inheritance pattern of mutagenesis at Bn JAG.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 Bn IND 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 Bn ALC,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
REFERENCES
APPENDIX
ACKNOWLEDGEMENTS
CURRICULUM VITAE
PUBLICATION FROM DOCTORAL THESIS
【参考文献】：
期刊论文
[1]Genome editing opens a new era of genetic improvement in polyploid crops[J]. Qamar U.Zaman,Chao Li,Hongtao Cheng,Qiong Hu. The Crop Journal. 2019(02)
[2]Rapeseed research and production in China[J]. Qiong Hu,Wei Hua,Yan Yin,Xuekun Zhang,Lijiang Liu,Jiaqin Shi,Yongguo Zhao,Lu Qin,Chang Chen,Hanzhong Wang. The Crop Journal. 2017(02)
[3]油菜抗裂角性鉴定方法的改进及试验[J]. 彭鹏飞,李云昌,梅德圣,刘道敏,付丽,王会,桑世飞,陈玉峰,胡琼. 农业工程学报. 2013(21)
[4]甘蓝型油菜抗裂角品种(系)的筛选与分析[J]. 文雁成,傅廷栋,涂金星,马朝芝,沈竞雄,张书芬. 作物学报. 2008(01)
本文编号：3587428
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