蜜蜂球囊菌（Ascosphaera apis）致病基因的CRISPR、CRISPR/Cas9编辑及致病机理研究

发布时间：2024-03-31 08:39

　　蜜蜂是一种高度社会化的昆虫,具有很高的经济价值和生态作用。目前,由于营养不足、自然栖息地的丧失、螨虫的侵袭、遗传多样性的丧失、杀虫剂和特异的疾病等原因,全世界饲养的蜜蜂蜂群正面临着严重的威胁,蜂群数量正在急剧下降。白垩病是由昆虫病原真菌蜜蜂球囊菌(Ascosphaera apis)引起的一种世界性的蜜蜂病害,也是造成全世界,尤其是中国养蜂业重大损失的重要原因。由于白垩病引起蜂群数量的大量减少,以及随之带来的巨大经济损失,研究人员开始寻找防治蜜蜂疾病的新策略,其中包括对致病菌致病机制的分子生物学研究。目前,基因工程技术是该研究的首选技术手段,即通过发现致病基因可能的作用机制,对白垩病进行生物防控。基于此,根据实验室的前期工作基础,本研究采取CRISPR/Cas9基因编辑技术对蜜蜂球囊菌的4个基因功能进行了研究,为今后开发防控蜜蜂白垩病新方法,提高蜜蜂的健康水平奠定基础。主要研究结果:1.从中国3个省的4个不同蜂场收集了蜜蜂球囊菌菌株4个,采用ITS序列测序方法验证了物种特异性,梯度培养的方法明确了蜜蜂球囊菌菌株对潮霉素B的敏感浓度为25μg/ml。2.在前期研究的基础上,选择了4个与蜜蜂...

【文章页数】：126 页

【学位级别】：博士

【文章目录】：
摘要
Abstract
Abbreviations
Chapter 1.General introduction
    1.1 Honeybees and their importance
        1.1.1 Honey bees' health
        1.1.2 Chalkbrood disease
        1.1.3 Control measures of Ascosphaera apis
        1.1.4 The pre-CRISPR/Cas9 gene editing era: REMI,HR,ZFNs,TALENs
        1.1.5 The new era of genetic engineering,the CRISPR/Cas9
        1.1.6 CRISPR/Cas9 genome editing tool working mechanism
        1.1.7 DSB repair pathways for CRISPR/Cas9 in filamentous fungi
    1.2 Multiplex genome editing with CRISPR/Cas9 system
    1.3 Delivery of Cas9 & gRNA
    1.4 Research attempts done on Ascosphaera apis
        1.4.1 Genetic engineering by Restricted Enzyme-Mediated Integration method
        1.4.2 Transcriptome analysis
    1.5 Objectives
Chapter 2.Isolation of Ascosphaera apis and identification of target genes forCRISPR/Cas9 gene editing
    2.1 Introduction
        NAD(P) binding domain protein (AAPI11758)
        Versicolorin reductase gene (StcU-2)
        Sterigmatocytin 8-O-methyl transferase (OmtA) (AAPI10160),and Super killer protein3 (Ski3)(AAPI15770) genes
    2.2 Materials & methods
        2.2.1 Samples
        2.2.2 Equipment
        2.2.3 Materials and reagents
        2.2.4 Methods
            2.2.4.1 Determining of A. apis hygromycin B antibiotic resistance level
            2.2.4.2 Fungal genomic DNA extraction
            2.2.4.3 Screening of A. apis ITS, and target genes to be edited
                Primer designing and synthesis
            2.2.4.4 Specific target genes and ITS amplification
    2.3 Results
        2.3.1 Hygromycin B resistance
        2.3.2 Assessment of fungal genomic DNA quality and quantity
        2.3.3 Ascosphaera apis genes to be edited
        2.3.4 Targeted gene DNA sequences
    2.4 Discussion
    2.5 Conclusion
Chapter 3.Pathogenicity and sporulation related gene editing of Ascosphaera apisusing CRISPR/Cas9
    3.1 Introduction
    3.2 Materials and methods
        3.2.1 Media and reagents preparation
        3.2.2 Equipment
        3.2.3 Materials and reagents
        3.2.4 Isolation and purification of protoplasts
        3.2.5 Regeneration and growth of protoplasts
        3.2.6 Effects of PEG4000 concentration on protoplast transformation
        3.2.7 Single guide RNA/Cas9-all-in-one Plasmid DNA construction, PEG-mediatedprotoplast transformation, and screening
        3.2.8 Confirming exon regions of the knocked out target genes
        3.2.9 Verification of mutants through fluorescence laser scanning microscopy, andsequence analysis
    3.3 Results
        3.3.1 Isolation and purification of protoplasts
        3.3.2 Regeneration and growth of protoplasts
        3.3.3 Effects of PEG4000 concentration on protoplast transformation
        3.3.4 CRISPR/Cas9 gene edition and mutant selection
        3.3.5 Verification of transformants
    3.4 Discussion
    3.5 Conclusion
Chapter 4.Sporulation, and in vitro-pathogenicity of CRISPR/Cas9 gene editedAscosphaera apis mutants to honeybee larvae
    4.1 Introduction
    4.2 Materials and methods
        4.2.1 Research material
        4.2.2 Media and reagents preparation
        4.2.3 Microscopic analysis of sporulation and mating tests
        4.2.4 In vitro larval rearing and pathogenicity bioassay
        4.2.5 Statistical Analysis
    4.3 Results
        4.3.1 Microscopic analysis and sporulation
        4.3.2 Larval infection rate and mortality percentages
    4.4 Discussion
    4.5 Conclusion
Chapter 5.General conclusion
References
Appendix
Acknowledgements
Author Resume



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