在維管植物中,木質(zhì)素是次生細(xì)胞壁中含量僅次于纖維素的生物大分子,在植物應(yīng)力響應(yīng)、機(jī)械支持和水分傳輸中起著重要作用。木質(zhì)素生物合成相關(guān)基因的表達(dá)、演化和功能研究為研究植物次生代謝通路的協(xié)調(diào)進(jìn)化提供了一個(gè)極好的模型,同時(shí)對秸稈的理化性質(zhì)的遺傳改良具有重要的意義。CRISPR/Cas9基因組編輯技術(shù)因其兼具高效、精準(zhǔn)、易用性和通用性等優(yōu)點(diǎn)。本研究分析了水稻木質(zhì)素單體合成途徑相關(guān)基因,利用CREP數(shù)據(jù)庫發(fā)現(xiàn)了27個(gè)表達(dá)量較高的基因,從中選取了肉桂醇脫氫酶基因CAD1、肉桂酸4-羥化酶基因C4H2、羥基肉桂酰轉(zhuǎn)移酶HCT1和HCT2這4個(gè)基因作為本研究的對象。利用CRISPR/Cas9系統(tǒng)進(jìn)行了編輯,目前已經(jīng)通過農(nóng)桿菌介導(dǎo)的成功獲得了相關(guān)的轉(zhuǎn)基因陽性單株,并對具體的編輯位點(diǎn)進(jìn)行了測序確認(rèn),獲得了目標(biāo)基因編輯的材料,為研究和改良木質(zhì)素單體合成途徑建立了基礎(chǔ)。同時(shí),本研究還對這些基因編碼的CAD1、C4H2、HCT1和HCT2蛋白質(zhì)的3 D結(jié)構(gòu),以及禾本科植物中的同源蛋白進(jìn)行了比較研究和聚類分析。該研究有望通過木質(zhì)素單體H、S和G配比的改造,為研究水稻植株的農(nóng)藝適應(yīng)性、秸稈的理化性質(zhì)的改變和秸稈的綜... 

【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

【文章頁數(shù)】：105 頁

【學(xué)位級(jí)別】：碩士

【文章目錄】：
摘要
ABSTRACT
ABBREVIATION
1 INTRODUCTION
    1.1 Literature review
        1.1.1 Rice as a Model Plant
        1.1.2 Lignin and Lignocellulosic Biomass Conversion
        1.1.3 The Lignin Monolignol Biosynthetic Pathway
        1.1.4 The Recent Developments in Transgenic Technologies and CRISPR/Cas9 Gene-Editing System
    1.2 Research Objectives
2 MATERIAL AND METHOD
    2.1 Selection of Genes
    2.2 Designing sgRNA
    2.3 Vector Construction
        2.3.1 Primer Renaturation
        2.3.2 CRISPR Vector Restriction Digestion
        2.3.3 DNA and Vector Ligation
    2.4 Transform Escherichia coli DH5α Competent Cells
        2.4.1 Preparation of DH5α Competent Cells
        2.4.2 Transforming DH5α Competent Cells
        2.4.3 LB-Medium
    2.5 Selection of Positive Colonies
        2.5.1 PCR Protocol
        2.5.2 Sanger Sequencing
    2.6 Sub-Clone Vector into Agrobacterium
        2.6.1 Freeze–Thaw Method
    2.7 Rice Transformation
        2.7.1 Induction of Calli
        2.7.2 Preparation of Calli and Agrobacterium
        2.7.3 Infection of Callus with Agrobacterium
    2.8 Detection of Positive Plants
        2.8.1 DNA Extraction
        2.8.2 CTAB Extraction Buffer
    2.9 Software and Websites Used
3 RESULTS AND ANALYSIS
    3.1 Collection of Protein Sequences and Alignment
    3.2 Identities of Genes with their Homologous Proteins
    3.3 Multiple Sequence Alignment and Phylogenetic Tree
    3.4 3D Proteins Structures Prediction
    3.5 Identification of Unique Sites in Proteins to Design sgRNA
    3.6 Designed sgRNA
    3.7 Vector Construction
    3.8 Identification of Positive E. coli Colonies and Sequencing Results
    3.9 Sub-Cloning of Plasmid into Agrobacterium tumefaciens
    3.10 T0 Generation Mutants
    3.11 Mutations Observed in T0 Generation
    3.12 Phenotypes Observed in Mutant Plants
    3.13 Mutants Protein Sequence Predictions and Alignment
4 DISCUSSION
    4.1 Some Remaining Issues
REFERENCES
Acknowledgements


【參考文獻(xiàn)】：
期刊論文
[1]Genetic Engineering of Energy Crops: A Strategy for Biofuel Production in China[J]. Guosheng Xie1 and Liangcai Peng1,2 1National Key Laboratory of Crop Genetic Improvement,Biomass and Bioenergy Research Centre,and College of Plant Sciences and Technology,Huazhong Agricultural University,Wuhan 430070,China 2College of Life Sciences and Technology,Huazhong Agricultural University,Wuhan 430070,China.  Journal of Integrative Plant Biology. 2011(02)



本文編號(hào)：3438273