Identification of Novel Nematicidal Crystal Proteins and Hos

发布时间：2020-12-25 02:42

　　苏云金芽胞杆菌（Bacillus thuringiensis,Bt）是一种昆虫病原菌,其特征是在形成芽胞的同时会产生由Cry蛋白组成的伴胞晶体,这些伴胞晶体对多种农业害虫具有毒杀活性。因此,Bt成为用于微生物农药最成功的微生物,此外,Bt的Cry蛋白基因也可以通过转基因作物的方式应用于农业上重要昆虫的防治。目前关于Bt Cry蛋白防治昆虫的研究相对较多,然而对于Cry蛋白防治线虫的研究还很有限。植物寄生线虫对农作物造成重大的损失,因此需要寻找对线虫具有较高毒性和较广谱的新型Cry蛋白。基于此,本研究集中于分离新菌株并使用基因组分析的方法来发掘新型Cry蛋白基因的工作。主要研究内容如下:1)通过100个Bt基因组分析发现在20%的Bt基因组中存在cry基因片段化的现象,通过对其中一个片段化基因cry5B-LN的功能研究发现该片段化基因与另一 Cry蛋白Cry5Ba的C端编码序列融合表达时,可以形成典型的伴胞晶体,同时该融合晶体蛋白可以造成线虫肠道上皮细胞连接的破坏,进而表现出对线虫具有毒杀活性,本研究结果揭示了片段化的cry基因可以作为潜在的具有活性的毒素资源。晶体蛋白典型的N端doma... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：129 页

【学位级别】：博士

【文章目录】：
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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