柑橘黄龙病菌致病相关基因表达、遗传多样性及传播特性的初步研究

发布时间：2021-11-01 22:16

　　柑橘黄龙病（HLB）是影响全球柑橘产业发展的最严重病害之一。目前该细菌尚无法获得离体纯培养,严重阻碍了其生物学特性、遗传变异和致病分子机制的研究。HLB菌全基因组序列的公布,为其致病机制研究提供了理论参考。在本研究中,为了探讨CLas致病的可能分子机制,我们对假定致病相关效应子基因在不同症状样本中的表达水平,以及柑橘中部分防卫相关基因进行了表达特性分析。另外对巴基斯坦样品中的黄龙病菌进行基于Cthr基因的遗传多样性分析。同时对利用菟丝子从柑橘向长春花和烟草传播CLas进行了研究,获得的主要结果如下:1.致病相关效应子基因表达分析:我们从中国主要的柑橘产区,包括江西、云南、海南等地,收集了表现不同症状的样品。运用q RT-PCR分析了9个效应子基因在均匀黄化（Y）、斑驳黄化（BM）、脉凸（VK）和缺锌黄化（ZD）症状中的表达特性。结果如下:CLas₀3230、CLas₀4025和CLas₀4560在VK症状中表达水平较高,而CLas₀2075、CLas₀2145、CLas

【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】：87 页

【学位级别】：硕士

【文章目录】：
Abstract
摘要
Abbreviation
Chapter1 Literature Review
    1.1 Introduction
    1.2 Symptoms of Huanglongbing and its Impact on Orange Trees
    1.3 Classification and distribution of pathogens
    1.4 Host plant-pathogen interactions
    1.5 Genomics of HLB bacteria
    1.6 Techniques for the detection of HLB bacteria
        1.6.1 Biological assay
        1.6.2 Microscopic techniques
        1.6.3 Molecular techniques
        1.6.4 Spectroscopic and imaging technique
        1.6.5 Profiling of plant volatile organic compounds for disease identification
        1.6.6 Isothermal amplification combined with a lateral flow dipstick for rapidand sensitive detection of Candidatus Liberibacter
    1.7 Transient expression of Candidatus Liberibacter asiaticus effectors
    1.8 Collagen-triple helix gene
        1.8.1 Triple-helix structure and stability
        1.8.2 Formation of higher order structures
        1.8.3 Bacterial collagens that are known to form a triple helix structure
    1.9 Aim and scope of this study
Chapter2 Gene expression analysis of putative pathogenesis-related genes of CandidatusLiberibacter asiaticus
    2.1 Introduction
    2.2 Material and methods
        2.2.1 Plant materials
        2.2.2 Primers used in this study
        2.2.3 Plant total genomic DNA extraction
        2.2.4 Plant total RNA extraction
        2.2.5 Preparation of c DNA
        2.2.6 RT-PCR reaction
        2.2.7 q RT-PCR reaction
    2.3 Result and Analysis
        2.3.1 Gene expression analysis by RT-PCR
        2.3.2 Gene expression analysis of effectors of CLas by q RT-PCR
    2.4 Discussion
    2.5 Conclusion
Chapter3 Genetic diversity analysis of Pakistan samples based on Cthr gene
    3.1 Introduction
    3.2 Material and methods
        3.2.1 Plant samples
        3.2.2 Plant total genomic DNA extraction
        3.2.3 Primers and PCR assays
        3.2.4 Recovery and purification of PCR products
        3.2.5 Ligation of target DNA fragments
        3.2.6 Recombinant heat shock transformation
        3.2.7 Phylogenetic analysis
    3.3 Results and analysis
        3.3.1 Identification of Cthr gene in CLas Psy62 genome
        3.3.2 PCR identification of Cthr gene
        3.3.3 Cthr gene sequence analysis
        3.3.4 Comparison of Cthr gene between Pakistan and representative isolates
        3.3.5 Phylogenetic analysis of CLas from Pakistan isolates based on Cthr gene
    3.4 Discussion
    3.5 Conclusion
Chapter4 CLas transmission from citrus to periwinkle and tobacco via dodder
    4.1 Introduction
    4.2 Materials and methods
        4.2.1 Preparation of plant materials
        4.2.2 Sample collection
        4.2.3 Plant total genomic DNA extraction
        4.2.4 PCR amplification
    4.3 Result
        4.3.1 CLas transmission via dodder from citrus to periwinkle
        4.3.2 CLas transmission via dodder from citrus to tobacco
        4.3.3 Identification of CLas in periwinkle and tobacco after transmission via dodder
    4.4 Discussion
    4.5 Conclusion
References
Acknowledgement


【参考文献】：
期刊论文
[1]应用PCR及Nested-PCR技术检测柑橘黄龙病病原研究[J]. 丁芳,易干军,王国平.  园艺学报. 2004(06)



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