蘇云金芽胞桿菌（Bacillus thuringiensis,Bt）是一種昆蟲(chóng)病原菌,其特征是在形成芽胞的同時(shí)會(huì)產(chǎn)生由Cry蛋白組成的伴胞晶體,這些伴胞晶體對(duì)多種農(nóng)業(yè)害蟲(chóng)具有毒殺活性。因此,Bt成為用于微生物農(nóng)藥最成功的微生物,此外,Bt的Cry蛋白基因也可以通過(guò)轉(zhuǎn)基因作物的方式應(yīng)用于農(nóng)業(yè)上重要昆蟲(chóng)的防治。目前關(guān)于Bt Cry蛋白防治昆蟲(chóng)的研究相對(duì)較多,然而對(duì)于Cry蛋白防治線(xiàn)蟲(chóng)的研究還很有限。植物寄生線(xiàn)蟲(chóng)對(duì)農(nóng)作物造成重大的損失,因此需要尋找對(duì)線(xiàn)蟲(chóng)具有較高毒性和較廣譜的新型Cry蛋白。基于此,本研究集中于分離新菌株并使用基因組分析的方法來(lái)發(fā)掘新型Cry蛋白基因的工作。主要研究?jī)?nèi)容如下:1)通過(guò)100個(gè)Bt基因組分析發(fā)現(xiàn)在20%的Bt基因組中存在cry基因片段化的現(xiàn)象,通過(guò)對(duì)其中一個(gè)片段化基因cry5B-LN的功能研究發(fā)現(xiàn)該片段化基因與另一 Cry蛋白Cry5Ba的C端編碼序列融合表達(dá)時(shí),可以形成典型的伴胞晶體,同時(shí)該融合晶體蛋白可以造成線(xiàn)蟲(chóng)腸道上皮細(xì)胞連接的破壞,進(jìn)而表現(xiàn)出對(duì)線(xiàn)蟲(chóng)具有毒殺活性,本研究結(jié)果揭示了片段化的cry基因可以作為潛在的具有活性的毒素資源。晶體蛋白典型的N端doma... 

【文章來(lái)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)】：129 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
ABSTRACT
中文摘要
ABBREVIATION
Chapter 1: Genome wide analysis of Bacillus thuringiensis reveals partial genescould be potential source of functional toxins
    1.INTRODUCTION
        1.1 Bacillus thuringiensis
            1.1.1 The Bacillus cereus group complex
            1.1.2 B.thuringiensis virulence factors
            1.1.3 B.thuringiensis insecticidal crystal protein
        1.2 Split toxin and partial genes
        1.3 Structural and functional relationship
        1.4 Mode of action
            1.4.1 Solubilisation and proteolytic activati
            1.4.2 Receptor binding
            1.4.3 Bravo-Soberon model
            1.4.4 The "ping-pong" sequential binding model
            1.4.5 Zhang-Bulla model
        1.5 Plant parasite nematodes and B.thuringiensis
            1.5.1 C.elegans as a model model oranism
        1.6 Molecular evolution of B.thuringiensis
        1.7 Aim and objective
    2.MATERIALS AND METHOD
        2.1 Bacterial strains
            2.1.1 Culture medium
            2.1.2 Standard polymerase chain reaction （PCR）
            2.1.3 PCR using thermostable polymerase
            2.1.4 Purification of PCR products
            2.1.5 Construction of recombinant plasmids
            2.1.6 Purification of plasmid DNA
            2.1.7 Sequencing of plasmid inserts
        2.2 Culture and maintenance of C.elegans
            2.2.1 Nematodes bioassays
            2.2.2 Spore and crystal preparation
            2.2.3 Protein separation by polyacrylamide gel electrophoresis
            2.2.4 Western blot analysis
            2.2.5 Dot blot analysis
            2.2.6 Scanning Electron Microscopy （SEM）
            2.2.7 Light microscopy
        2.3 Bioinformatics methods
            2.3.1 Mining of Cry protein from B.thuringiensis
            2.3.2 Software and databases used
            2.3.3 Phylogenetic analysis
            2.3.4 Statistical analysis
    3 .RESULTS AND ANALYSIS
        3.1 Partial genes are widely distributed in the genomes of B.thuringiensis
        3.2 Bioinformatics analysis of Cry5B-like N-terminal protein
        3.3 Identification of Cry5B like N-terminal, unable to expression in BMB171 andwild type B.thuringiensis C15
        3.4 C-terminal helps in cry stallization of Cry5B-like N-terminal
        3.5 Hybrid N+C protein is toxic to nematodes
        3.6 N+C hybrid protein cause pathogenesis through intestine infection
    4.DISCUSSION AND CONCLUSION
Chapter 2:Identification of Novel Cry5Fa Crystal protein with Nematicidal Activity
    1.INTRODUCTION
        1.1 Plant parasite nematodes a major threat to agriculture
        1.2 Nematicidal activity of Cry toxins
            1.2.1 Search for novel cry toxin having wider toxicit
        1.3 Objective and significance
    2.MATERIALS AND METHODS
    3.RESULTS AND ANALYSIS
        3.1 Genome screening for Novel Cry toxins
        3.2 Cry5Fa cause the pathogenesis towards C.elegans
        3.3 Cry5Fa and Cry5Ba showed different activity against mutant C.elegans
Chapter 3: Hydralysin, Aerolysin like protein as a virulence factor
    1.INTRODUCTION
        1.1 Presence of aerolysin like virulance factors in B.thuringiensis
    2.MATERIALS AND METHODS
    3.RESULTS AND ANALYSIS
        3.1 Bioinformatics analysis of hydralysin
        3.2 Cloning and Expression of Hydralysin protein
        3.3 Hydralysin synergistically enhance the activity of Cry5Ba
    4.CONCLUSION
REFERENCE
Published papers and Awards
APPENDIX
ACKNOWLEDGEMENT



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