苏云金芽孢杆菌的重要功能基因

论文摘要

害虫抗性问题已经成为苏云金芽孢杆菌的重要研究课题。不少研究者在寻找新的苏云金芽孢杆菌主要杀虫基因—cry。PCR-RFLP技术已被证实可以快速分析鉴定苏云金芽孢杆菌菌株的cry和cyt基因型。本研究在继承前人已经成功建立的cry1-cry4、cry7、cry9和cry10鉴定体系基础上，发展了cry5、cry6、cry8和cry11的PCR-RFLP鉴定体系，进而采用上述PCR-RFLP技术鉴定了苏云金芽孢杆菌6株标准菌株和30株武夷山分离株的cry1-cry11基因类型。结果发现，88.9％、61.1％和2.8％的菌株分别含有cry1、cry2和cry11类基因，从供试菌株中检测到22种cry基因亚型（cry1Aa、cry1Ab、cry1Ac、cry1Ad、cry1Ba、cry1Cb、cry1Db、cry1Ea、crylEb、cry1Fa、cry1Fb、cry1Ga、cry1Gb、cry1Ka、cry1La、cry1Ia、cry1Ib、cry2A6、cry2Ac、cry2Ad、cry2Ae和cry11Aa）。然而，所有供试菌株均未检测到cry3、cry4、cry5、cry6、cry7、cry8、cry9和cry10基因型。第一代转基因棉花主要含有cry1Ac。然而多种害虫已经对苏云金芽孢杆菌产生抗性，而鳞翅目重要害虫小菜蛾在田间条件下已经对Cry1Ac产生了抗性。由于cry2Ab与cry1Ac序列同源性较低，两者不大可能产生交互抗性，国外学者已经以cry2Ab基因为基础获得第二代转基因棉花。目前，苏云金芽孢杆菌内毒素基因国际命名委员会网站（http://www.lifesci.sussex.ac.uk/home/NeilCrickmore/Bt/）共报道了7个cry2Ab基因和1个cry2Ad基因。本研究从武夷山分离株WB2和WB10中分别克隆到一个cry2基因（已被苏云金芽孢杆菌内毒素基因国际命名委员会命名为cry2Ab8和cry2Ad2）。cry2Ab8全长1902bp，编码633aa，所推导的氨基酸序列与已知的Cry2Ab基因氨基酸序列同源性最高达99.5％，与所有已知的Cry2Ab相比存在3个氨基酸的差异（Ala411Val、Ile629Leu和Ser630Pro）。cry2Ad2全长1902bp，编码633aa，所推导的氨基酸序列与Cry2Ad1氨基酸同源性最高（99.4％），有4个氨基酸的差异（Asn10Thr、Ser354Pro、Ile629Leu和Ser630Pro）。 苏云金芽孢杆菌自1901年被发现以来，一直缺乏有效的抗病基因资源。最近，研究人员发现在苏云金芽孢杆菌中存在一种抗病基因aiiA，其表达产物AiiA能减弱欧文氏菌导致的软腐病危害。为了有效利用这种新型抗病基因资源，本研究利用PCR技术初步研究了aiiA基因在芽孢杆菌中的分布情况。结果表明：在5株苏云金芽孢杆菌标准菌株、29株苏云金芽孢杆菌武夷山分离株、3株蜡质芽孢杆菌标准菌株和1株枯草芽孢杆菌分离株中均含有aiiA基因。将来自3株苏云金芽孢杆菌武夷山分离株、2株蜡质芽孢杆菌标准菌株和BS-1的aiiA进行分子克隆。在线核苷酸序列BLAST

论文目录

• Statement in Chinese
• Contents
• List of Figures
• List of Tables
• List of Abbreviations
• Abstract in Chinese
• Abstract in English
• Chapter 1 Bacillus thuringiensis: from gene to genomics to commercialization
• 1.1 Introduction
• 1.2 B. thuringiensis research and development
• 1.2.1 Major events in the progress of B. thuringiens
• 1.2.2 Key bioactive elements of B. thuringiensis
• 1.2.2.1 δ-endotoxins
• 1.2.2.2 β-exotoxin
• 1.2.2.3 Chitinase
• 1.2.2.4 Vegetative insecticidal proteins
• 1.2.2.5 Zwittermicin A
• 1.2.2.6 non-Cyt inclusion proteins against cancer cells
• 1.2.2.7 S-layer proteins
• 1.2.3 Mode of action
• 1.2.3.1 Tridimensional structure of Cry toxins and their functions
• 1.2.3.2 Helper proteins （Molecular chaperons）
• 1.2.3.3 Plasmid transfer
• 1.2.4 cry gene expression and regulation
• 1.2.5 Transgenic expression of B. thuringiensis toxin genes
• 1.2.5.1 Genetically modified microorganisms
• 1.2.5.2 Fermentation study
• 1.2.5.3 Transgenic plants
• 1.2.6 Registered biopesticides based on B. thuringiensis in P R China and U.S
• 1.3 Problems & solutions
• 1.3.1 Relationship between B. thuringiensis and B. cereus
• 1.3.2 Safety assessment or B. thuringiensis toxins
• 1.3.2.1 Effects on soil
• 1.3.2.2 Effects on water
• 1.3.2.3 Effects on plants
• 1.3.2.4 Effects on non-target microbes and invertebrates
• 1.3.2.5 Effects on vertebrates
• 1.3.2.6 Effects on food
• 1.3.3 Comparative sensitivity to UV radiation
• 1.3.4 Resistance and insect resistance management
• 1.3.4.1 Mechanism of resistance
• 1.3.4.2 Insect resistance management
• 1.4 Progress in B. thuringiensis
• 1.4.1 Screening and identification of B. thuringiensis strains
• 1.4.2 Identification of ICPs genes
• 1.4.3 Toxin receptors
• 1.4.4 Quorum-sensing
• 1.4.5 Secretion of metabolites
• 1.4.6 B. thuringiensis phages
• 1.4.7 Functional genomics
• 1.4.7.1 Genome sequencing
• 1.4.7.2 Identification of novel functional genes
• 1.4.7.3 Transposable elements
• 1.4.7.4 Proteomics analysis
• 1.4.7.5 Transcriptomics analysis
• 1.5 Conclusion
• 1.6 Rationale and significance
• Chapter 2 PCR-RFLP screening of cry gene profiles in B. thuringiensis and characterization of two novel cry2-type genes
• 2.1 Introduction
• 2.2 Materials and methods
• 2.2.1 Bacterial strains
• 2.2.2 Preparation of plasmid DNA
• 2.2.3 PCR-RFLP screening of cry genes
• 2.2.4 PCR amplification of cry2-type genes
• 2.2.5 Molecular cloning and sequencing of cry2-type genes
• 2.2.6 Data analysis
• 2.2.7 B. thuringiensis toxin nomenclature
• 2.2.8 Nucleotide sequence accession numbers
• 2.3 Results
• 2.3.1 Identification of cry genes classes from B. thuringiensis
• 2.3.2 Diversity of cry gene combinations of B. thuringiensis isolates
• 2.3.3 Cloning and sequence analysis of cry2Ab8
• 2.3.4 Cloning and sequence analysis of cry2Ad2
• 2.4 Discussion
• Chapter 3 Cloning and characterization of aiiA from Bacillus: evidence for the existence of an AHL-lactonase gene in B. subtilis BS-1
• 3.1 Introduction
• 3.2 Materials and methods
• 3.2.1 Bacterial strains and culture conditions
• 3.2.2 DNA preparation, primers and PCR amplification
• 3.2.3 Molecular cloning and sequencing
• 3.2.4 Sequence analysis
• 3.2.5 Three-dimensional structure prediction
• 3.2.6 Expression of the recombinant GST-AiiA fusion protein
• 3.2.7 Biological activity assay of BS-1 and its GST-AiiA fusion protein
• 3.2.8 Nucleotide sequence accession numbers
• 3.3 Results
• 3.3.1 Detection of aiiA in B. thuringiensis, B. cereus and B. subtilis
• 3.3.2 Cloning and sequence analysis of the aiiA gene
• 3.3.3 3-D structure prediction of putative AiiA from BS-1
• 3.3.4 BS-1 and its GST-AiiA fusion protein suppressed the virulence of E, carotovora var. carotovora Dya
• 3.4 Discussion
• Chapter 4 Identification, distribution pattern of IS231 elements in B. thuringiensis and their phylogenetic analysis
• 4.1 Introduction
• 4.2 Materials and methods
• 4.2.1 Bacterial strains and plasmids
• 4.2.2 DNA sample preparation and PCR conditions
• 4.2.3 IS231-like elements detection
• 4.2.4 Molecular cloning
• 4.2.5 Data and phylogenetic analysis
• 4.2.6 Nucleotide sequence accession numbers
• 4.3 Results
• 4.3.1 Occurrence of IS231 elements
• 4.3.2 Data analysis of novel IS231
• 4.3.3 Phylogenetic analysis
• 4.4 Discussion
• Chapter 5 Identification of genes differentially expressed during the development of B. thuringiensis
• 5.1 Introduction
• 5.2 Materials and methods
• 5.2.1 Bacterial strains and plasmids
• 5.2.2 RNA preparation
• 5.2.3 RAP-PCR
• 5.2.4 Isolation, cloning of RAP-PCR fragments
• 5.2.5 DNA sequencing
• 5.2.6 Confirmation of differential gene expression
• 5.2.7 Nucleotide sequence accession numbers
• 5.3 Results
• 5.3.1 Growth process of B. thuringiensis strain 8010
• 5.3.2 RAP-PCR analysis
• 5.3.3 Verification of differential gene expression by specifically primed RT-PCR
• 5.3.4 Characterization of the growth-regulated genes
• 5.4 Discussion
• Chapter 6 Concluding remarks
• References
• Appendices
• Acknowledgements