蜜蜂是一種高度社會(huì)化的昆蟲,具有很高的經(jīng)濟(jì)價(jià)值和生態(tài)作用。目前,由于營養(yǎng)不足、自然棲息地的喪失、螨蟲的侵襲、遺傳多樣性的喪失、殺蟲劑和特異的疾病等原因,全世界飼養(yǎng)的蜜蜂蜂群正面臨著嚴(yán)重的威脅,蜂群數(shù)量正在急劇下降。白堊病是由昆蟲病原真菌蜜蜂球囊菌(Ascosphaera apis)引起的一種世界性的蜜蜂病害,也是造成全世界,尤其是中國養(yǎng)蜂業(yè)重大損失的重要原因。由于白堊病引起蜂群數(shù)量的大量減少,以及隨之帶來的巨大經(jīng)濟(jì)損失,研究人員開始尋找防治蜜蜂疾病的新策略,其中包括對(duì)致病菌致病機(jī)制的分子生物學(xué)研究。目前,基因工程技術(shù)是該研究的首選技術(shù)手段,即通過發(fā)現(xiàn)致病基因可能的作用機(jī)制,對(duì)白堊病進(jìn)行生物防控�；诖�,根據(jù)實(shí)驗(yàn)室的前期工作基礎(chǔ),本研究采取CRISPR/Cas9基因編輯技術(shù)對(duì)蜜蜂球囊菌的4個(gè)基因功能進(jìn)行了研究,為今后開發(fā)防控蜜蜂白堊病新方法,提高蜜蜂的健康水平奠定基礎(chǔ)。主要研究結(jié)果:1.從中國3個(gè)省的4個(gè)不同蜂場收集了蜜蜂球囊菌菌株4個(gè),采用ITS序列測序方法驗(yàn)證了物種特異性,梯度培養(yǎng)的方法明確了蜜蜂球囊菌菌株對(duì)潮霉素B的敏感濃度為25μg/ml。2.在前期研究的基礎(chǔ)上,選擇了4個(gè)與蜜蜂...

【文章頁數(shù)】：126 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
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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