大豆(Glycinemax)是一種豆科作物,在豆類生產(chǎn)中較大范圍的商業(yè)應(yīng)用因此得以在全世界范圍內(nèi)廣泛種植。大豆油是人類食物的重要組成部分,并且是眾多工業(yè)生產(chǎn)應(yīng)用的原材料。脂質(zhì)合成和代謝是生命系統(tǒng)的主要活動(dòng),例如,三酰甘油(TAG)是儲(chǔ)存物質(zhì),而磷脂在生物膜的形成中發(fā)揮著至關(guān)重要的作用,而生物膜在植物早期發(fā)育和生長(zhǎng)中起著關(guān)鍵作用,同時(shí)磷脂作為食物成分也為人類提供了營(yíng)養(yǎng)來源。傳統(tǒng)的(育種)方法是通過調(diào)控油脂合成途徑中的關(guān)鍵基因來實(shí)現(xiàn)的。然而這種方法的局限性在于只能夠調(diào)控脂肪酸合成、降解過程特定的某一個(gè)基因。克服這一缺點(diǎn)的方法,就是通過操縱潛在的轉(zhuǎn)錄因子,激活調(diào)控油脂合成復(fù)合途徑中的多個(gè)基因。本研究的主要目的,即通過操縱ABSCISIC ACID-INSENSITIVE3(ABI3),FUSCA3(FUS3)和LEAFYCOTYLEDON2(LEC2;AFL)等B3結(jié)構(gòu)域的轉(zhuǎn)錄因子和HAP3結(jié)構(gòu)域轉(zhuǎn)錄因子LEC1提高植物組織中的油份;這些轉(zhuǎn)錄因子在種子發(fā)育以及種子中代謝物諸如蛋白質(zhì)、碳水化合物和脂質(zhì)儲(chǔ)存過程中發(fā)揮重要作用。我們克隆了大豆中的這些轉(zhuǎn)錄因子,通過GATEWAY體系構(gòu)建到p B2GW...

【文章頁數(shù)】：113 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER Ⅰ. LITERATURE REVIEW
    1.1 General background
    1.2 Lipid biosynthesis
        1.2.1 FA biosynthesis
        1.2.2 Galactolipid biosynthesis in the chloroplast
        1.2.3 Neutral and polar lipids biosynthesis in the endoplasmic reticulum
    1.3 TAG storage in oil body
    1.4 The transporters involved in lipid degradation and beta oxidation
    1.5 Cross-membrane lipid transport and trafficking
        1.5.1 Flip-flop movements
        1.5.2 Plastid-ER FA transport
        1.5.3 ER-Plastid phospholipid and FA transport
    1.6 Transporters required for surface lipid deposition
    1.7 Role of transcriptional regulation in lipid accumulation
        1.7.1 Leafy cotyledon 1 (LEC1)
        1.7.2 Leafy cotyledon 2 (LEC2)
        1.7.3 Abscisic acid insensitive (ABI3) and FUSCA3 (FUS3)
        1.7.4 Wrinkled 1 (WRI1)
        1.7.5 Dofs increase the total FA content in transgenic plants
        1.7.6 Role of Basic leucine zipper (b ZIPs) in lipid accumulation
        1.7.7 MYB TFs regulate biosynthesis of VLCFAs
        1.7.8 Negative regulators of oil biosynthesis
    1.8 Biotechnology tools for metabolic engineering of oil yield and composition
    1.9 Economical importance of soybean crop
    1.10 Aims and objectives of the current study
CHAPTER Ⅱ. MATERIALS AND METHODS
    2.1 Identification of the soybean LAFL gene and in silico analysis
    2.2 Phylogenetic analysis
    2.3 Gene cloning
    2.4 Gel electrophoresis and purification of PCR product
    2.5 Arabidopsis thaliana transformation
    2.6 Plant growth conditions and treatment
    2.7 RNA extraction
    2.8 Quantitative RT-PCR (q RT-PCR) analysis of gene expression
    2.9 Genomic DNA extraction
    2.10 Plasmid extraction
    2.11 Seed oil extraction
    2.12 Hairy root induction
    2.13 Hairy root oil extraction
    2.14 Histochemical staining for starch
    2.15 Spectrophotometric determination of starch
    2.16 Protein extraction
    2.17 Anthocynin measurement
    2.18 CDNA library construction for Illumina deep sequencing
    2.19 Statistic analysis
CHAPTER Ⅲ. RESULTS
    3.1 Identification of LAFL genes in Glycine max genome
    3.2 Expression of Gm LAFL during soybean seed development
    3.3 Expression in Arabidopsis thaliana mutants alters seed oil production and TAG composition
    3.4 Ectopic expression in Arabidopsis thaliana modifies seed oil production and TAG composition
    3.5 GmLAFL genes alters the chemical composition of seeds
    3.6 GmLEAFY ectopic expression increases total protein in Arabidopsis seeds
    3.7 GmLEAFY genes are responsible for embryonic-vegetative phase transition
    3.8 Ectopic expression in soybean hairy roots promotes TAG accumulation
    3.9 Transcriptome analysis of over expressed soybean hairy roots
CHAPTER Ⅳ. DISCUSSION
    4.1 Each member of LAFL-clade regulate TAG content and composition
    4.2 GmLEC2 regulates starch and protein metabolism
    4.3 GmLEC2 and GmFUS3 target key regulatory genes in oil biosynthesis
    4.4 GmLEC2 and GmFUS3 target key genes involved in FA and TAG biosynthesis
    4.5 Genes involved in TAG catabolism and lipid hydrolysis are regulated by GmLEC2 and GmFUS3
    4.6 GmFUS3 and GmLEC2 control the regulation of lipid transporters
CHAPTER Ⅴ. CONCLUSIONS
REFERENCES
APPENDIX A. LIST OF PRIMERS USED IN THIS STUDY
APPENDIX B. PREDICTED EXPRESSION OF LAFL GENES IN DIFFERENT TISSUES OF SOYBEAN IN PUBLIC DATABASE (PHYTOZOME)
APPENDIX C. PHYLOGENETIC ANALYSIS OF GMFUS3 AND GMLEC2 TARGETS IN G.MAX AND THEIR ORTHOLOGS IN OTHER CROPS
APPENDIX D. LIST OF TRANSCRIPTION FACTORS AND TRANPORTERS REGULATED BY GMLEC2 AND GMFUS3 IN LIPID BIOSYNTHESIS PATHWAY
ACKNOWLEDGEMENT
PUBLICATIONS



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