棉花是世界上最重要的經(jīng)濟(jì)作物之一,棉花纖維是全球經(jīng)濟(jì)的基礎(chǔ)和紡織業(yè)的支柱,其他副產(chǎn)品可以作為牲畜飼料、棉籽油、肥料、紙張及其他消費(fèi)品,另外,棉花可以作為一種理想的植物,對(duì)基因組進(jìn)化、多倍體化和單細(xì)胞生物學(xué)等進(jìn)行不同的生物學(xué)研究。MYB轉(zhuǎn)錄因子包含一個(gè)MYB域,由52個(gè)氨基酸基序的4個(gè)不完全重復(fù)序列組成,該轉(zhuǎn)錄因子在保衛(wèi)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中起著重要作用。AtMYB44是一種多面轉(zhuǎn)錄因子,主要與擬南芥乙烯信號(hào)通路相關(guān)。APETALA2/乙烯反應(yīng)因子（AP2/ERF）是植物特異性轉(zhuǎn)錄因子（TFs）的一個(gè)超家族,它在調(diào)節(jié)植物器官發(fā)育,不同環(huán)境和生物脅迫的反應(yīng)中起著至關(guān)重要的作用。NB-ARC結(jié)構(gòu)域是一種調(diào)節(jié)程序性細(xì)胞死亡的STAND（信號(hào)轉(zhuǎn)導(dǎo)多個(gè)結(jié)構(gòu)域的ATP酶）蛋白,能夠與ATP結(jié)合進(jìn)行水解并誘導(dǎo)下游阻力信號(hào)傳導(dǎo)�；蚪M編輯（GE）是通過編輯不同生物的基因組序列從而改變生物特性的一種方法。近年來,從簡(jiǎn)單到復(fù)雜的基因組,人們已經(jīng)探索了各種各樣的基因編輯工具,CRISPR（成簇的有規(guī)律的間隔的短回文重復(fù)序列）是最新并且功能最為強(qiáng)大的一種編輯技術(shù),它的應(yīng)用徹底改變了科學(xué)界。CRISPR/CAS9是一種R... 

【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：157 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
Abstract
List of abbreviations
CHAPTER 1
    1 Introduction
        1.1 Cotton production and insect attack
            1.1.1 The interaction and defense of plants against insect stress
        1.2 Transcription factors(TFs)
            1.2.1 MYB transcription factors
                1.2.1.1 MYB44 gene
            1.2.2 APETALA2/Ethylene-Responsive Factor(AP2/ERF)gene
            1.2.3 Nucleotide-binding(NB)-ARC domain-containing disease resistanceprotein
        1.3 Whole genome sequencing to detect on and off-targets
        1.4 Genome editing techniques and their applications
            1.4.1 Genome editing
                1.4.1.1 The CRISPR/Cas system
                    1.4.1.1.1 The mechanism of the CRISPR/Cas9 system
                    1.4.1.1.2 Methodology for the screening of CRISPR/Cas9-induced mutant
                    1.4.1.1.3 The diverse applications of CRISPR/Cas9 system in major crops,Vegetables and fruits
        1.5 Aims of the study
CHAPTER 2
    2 Materials and methods
        2.1 Sequence analysis of GhMYB44 and GhAP2 and GhARC genes
        2.2 Subcellular localization of the GhMYB44 gene
        2.3 Computational sgRNA design and selection
        2.4 Vector construction for CRISPR/Cas9 and overexpression
        2.5 Plant material for Agrobacterium-mediated transformation in cotton
        2.6 Initiation,maintenance of embryogenic calli and plant regeneration
        2.7 Plant material,growth conditions and measurement of different agronomictraits
        2.8 DNA extraction and Southern blotting
        2.9 RNA extraction,RT-PCR,and qRT-PCR
        2.10 Tissue culturing and generation of homozygous transgenic lines
        2.11 On-target analysis of CRISPR/Cas9 edited plants
        2.12 Genomic DNA isolation and library construction for WholeGenome Sequencing
        2.13 Bioinformatics analysis for variant calling
        2.14 Genome-wide prediction off-target cleavage sites and detectiontheir mutations
        2.15 Analysis genetic variation of potential off-targets and PAMs between WTand cotton reference genome
        2.16 Data availability
CHAPTER 3
    3 RESULTS
        3.1 Characteristics of GhMYB44,GhAP2,and Gh ARC genes
        3.2 Knockout of GhMYB44,GhAP2,and GhARC genes and overexpression ofGhMYB44 gene in cotton
        3.3 Confirmation of GhMYB44,GhAP2,and GhARC transgenes and generationof homozygous lines
        3.4 Subcellular localization of GhMYB44 gene and Expression analysis ofGhMYB44,GhAP2,and GhARC genes
        3.5 Measurement of agronomic traits of GhMYB44,GhAP2,and GhARC genes
        3.6 Whole genome sequencing of CRISPR/Cas9-edited cotton plants,wild-typeand negative plants
        3.7 Detection of on-target mutations at the genome-wide scale
        3.8 Inherent genetic variation or/and somaclonal variation in Cas9-edited plants,wild-type,and negative plants
        3.9 Low frequency off-target mutations detected in Cas9-edited plants
        3.10 Genetic variation among cultivars can generate novel off-target sites
        3.11 Investigation of spontaneous mutations and the inheritance of Cas9-editedmutations
CHAPTER 4
    4 Discussion
Conclusion and future perspectives
REFERENCES
Appendices
Acknowledgements



本文編號(hào)：3540822