油菜角果在收獲時(shí)段易裂會(huì)造成嚴(yán)重的產(chǎn)量損失。油菜角果易裂與角果離區(qū)細(xì)胞降解密切相關(guān)。一些基因,諸如Bn JAG,Bn SHP,Bn IND和Bn ALC都參與到角果離區(qū)形成及發(fā)育中。為了提高油菜抗裂角性,利用CRISPR/Cas9系統(tǒng)將Bn JAG,Bn SHP,Bn IND和Bn ALC分別進(jìn)行敲除。對(duì)油菜中Bn JAG所有拷貝進(jìn)行敲除后,角果的大小及側(cè)生組織發(fā)育受到很大影響。整個(gè)角果成“愈傷”狀,沒(méi)有明顯的果瓣,隔膜和離區(qū)。此外,角果隔膜不能延伸到整個(gè)角果,隔膜兩側(cè)產(chǎn)生假種子,這些種子不能發(fā)育成熟。然而,當(dāng)只有Bn JAG.A08-NUB-Like（Bn JAG-A08）突變時(shí),隔膜發(fā)育完好,延伸到整個(gè)角果,將角果分成兩半。整個(gè)角果相對(duì)于野生型變短變粗。為了探討B(tài)n SHP基因功能,我們將Bn SHP1-157（Bn SHP1-A09突變）和Bn SHP4-484（四個(gè)Bn SHP基因突變系）雜交,產(chǎn)生五個(gè)Bn SHP突變系（Bn SHP5-184）。Bn SHP5-184中,離區(qū)木質(zhì)化層和離區(qū)分離層退化,角果SRI值為0.31顯著高于野生型（SRI=0.036）。此外,對(duì)Bn S... 

【文章來(lái)源】：中國(guó)農(nóng)業(yè)科學(xué)院北京市

【文章頁(yè)數(shù)】：115 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
DEDICATION
摘要
ABSTRACT
英文縮略表
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


【參考文獻(xiàn)】：
期刊論文
[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]油菜抗裂角性鑒定方法的改進(jìn)及試驗(yàn)[J]. 彭鵬飛,李云昌,梅德圣,劉道敏,付麗,王會(huì),桑世飛,陳玉峰,胡瓊.  農(nóng)業(yè)工程學(xué)報(bào). 2013(21)
[4]甘藍(lán)型油菜抗裂角品種(系)的篩選與分析[J]. 文雁成,傅廷棟,涂金星,馬朝芝,沈競(jìng)雄,張書芬.  作物學(xué)報(bào). 2008(01)



本文編號(hào)：3587428