大麗輪枝菌是一種土傳、植物性病原真菌,可以引起多種重要經(jīng)濟(jì)作物的枯萎死亡。一旦植物被感染后,尚無有效的防治措施。目前,大量的研究工作集中于病原菌毒力及致病關(guān)鍵基因的篩查。盡管農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法普遍用于外源基因的轉(zhuǎn)化,但是仍然需要一種更加快捷的轉(zhuǎn)化方法。本研究中,我們獲得了高質(zhì)量的大麗輪枝菌原生質(zhì)體用于外源基因的遺傳轉(zhuǎn)化。利用此體系,獲得了針對鐵還原酶跨膜元件前體3（FreB,VDAG₀6616）基因敲除突變體和回補體。隨后,對Fre B基因在病原菌鐵還原酶活性以及毒力方面的功能進(jìn)行了深入的研究。具體研究結(jié)果如下:1.利用崩潰酶消化并獲得了高質(zhì)量的原生質(zhì)體,在TB3液體培養(yǎng)基中再生效率可達(dá)65%。通過轉(zhuǎn)化方式（電擊和聚乙二醇）以及外源基因狀態(tài)（線性DNA和質(zhì)粒）對比發(fā)現(xiàn):在PEG介導(dǎo)的遺傳轉(zhuǎn)化中,每微克線性GFP表達(dá)盒可得到600個轉(zhuǎn)化子,每微克GFP質(zhì)粒可得到250個轉(zhuǎn)化子;電擊遺傳轉(zhuǎn)化中,每微克線性化GFP表達(dá)盒可得到29個轉(zhuǎn)化子,每微克GFP質(zhì)�？傻玫�24個轉(zhuǎn)化子。2.為探索小干擾RNA（siRNA）能否成功導(dǎo)入原生質(zhì)體并發(fā)揮基因沉默作用,針對上述研究獲... 

【文章來源】：中國農(nóng)業(yè)科學(xué)院北京市

【文章頁數(shù)】：86 頁

【學(xué)位級別】：博士

【文章目錄】：
摘要
abstract
Abbreviations
CHAPTER I INTRODUCTION
    1.1 Verticillium dahliae
        1.1.1 Life cycle of Verticillium dahliae
        1.1.2 Host range of Verticillium dahliae
        1.1.3 Symptoms caused by Verticillium dahliae
    1.2 Transformation of Verticillium dahliae
        1.2.1 Biolistic transformation
        1.2.2 Electroporation
        1.2.3 Agrobacterium tumefaciens-mediated transformation (ATMT)
        1.2.4 PEG-mediated transformation
    1.3 Ferric reductase transmembrane component 3 precursor
    1.4 Aims and objectives of the study
CHAPTER II MATERIALS AND METHODS
    2.1 Fungal growth and spores’ collection
    2.2 Protoplast isolation
    2.3 Regeneration of protoplast
    2.4 GFP plasmid and siRNAs
    2.5 PEG-mediated transformation
    2.6 Electroporation
    2.7 siRNA inhibition assay for GFP and Vta2 genes
    2.8 RNA extraction
    2.9 First strand cDNA synthesis
    2.10 qRT-PCR analysis of Vta2 expression level
    2.11 Genomic DNA isolation
    2.12 Phylogenetic analysis of FreB
    2.13 Plasmid construction for mutants’ generation
        2.13.1 PCR amplification of the respective fragments and genes
        2.13.2 Gel purification of PCR products by Gel purification kit
        2.13.3 Cloning the purified PCR product into T-vector
        2.13.4 Transformation into Escherichia coli (heat shock method)
        2.13.5 Construction of gene knockout fragment
        2.13.6 Construction of GFP tagged plasmid
        2.13.7 Construction of plasmid for complementation
        2.13.8 Extraction of plasmid
    2.14 PEG-mediated protoplast transformation for mutants’ generation
        2.14.1 Generation of FreB knockout mutants (ΔFreB)
        2.14.2 Generation of GFP disruption mutants
        2.14.3 Generation of FreB complementary mutants (ΔFreB-C)
    2.15 Growth of ΔFreB on different carbon sources
    2.16 Analysis of growth on media with different iron sources
    2.17 Ferric reductase assay
    2.18 Oxidative stress assay
    2.19 Pathogenicity assay
    2.20 Disease index
    2.21 Expression analysis of related genes
    2.22 Composition of media and buffers
CHAPTER III Building an efficient transformation system for Verticillium dahliae
    3.1 Introduction
    3.2 Results
        3.2.1 Isolation of protoplasts
        3.2.2 Regeneration of protoplast
        3.2.3 Observation of fluorescence from GFP expression
        3.2.4 GFP transformants selection and stability of the transgene
        3.2.5 Silencing of GFP gene in Vd-GFP strain with siRNAs
        3.2.6 Silencing of Vta2 gene
    3.3 Conclusion
CHAPTER IV Plasmid construction for gene deletion and complementation
    4.1 Introduction
    4.2 Results
        4.2.1 Phylogenetic analysis
        4.2.2 Generation of FreB mutants
        4.2.3 Generation of GFP tagged mutants
        4.2.4 Generation of FreB complementary mutants
    4.3 Conclusion
CHAPTER V Analysis of ΔFreB mutants
    5.1 Introduction
    5.2 Results
        5.2.1 Carbon sources utilization assay
        5.2.2 Comparing the growth of mutants on different iron sources
        5.2.3 Deletion of FreB gene impaired the surface ferric reductase activity
        5.2.4 Disruption of FreB gene resulted in increased susceptibility to oxidative stress54
        5.2.5 Deletion of FreB gene resulted in increased expression level of related genes..555.2.6 FreB deletion attenuates virulence of V. dahliae
        5.2.6 FreB deletion attenuates virulence of V. dahliae
    5.3 Conclusion
DISCUSSION
CONCLUSION
REFERENCES
ACKNOWLEDGEMENTS
CURRICULUM VITAE


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期刊論文
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