卵菌屬于假藻界,進化上與光合藻類相近。卵菌中有800個種,其中包括諸多植物病原菌,如疫霉屬與腐霉屬。在卵菌排名第五,引起巨大的經(jīng)濟與危害性。辣椒疫霉（P.capsici）是一種絲狀的植物病原卵菌,其寄主范圍廣泛,對茄屬、豆科、瓜果類植物均有極大的危害。真核生物通過受體蛋白激酶（RPKs）來感應(yīng)、感知與傳導(dǎo)來自細胞表面接收到的信號�；赗PKs的蛋白結(jié)域的不同,它們被分為不同的類別。在多個基因組中均發(fā)現(xiàn)富含亮氨酸重復(fù)的類受體蛋白激酶（LRR-RLKs）,它們具有影響生長、發(fā)育與對生物與非生物脅迫的免疫調(diào)節(jié)等多種生物學(xué)功能。我們在卵菌的基因組也發(fā)現(xiàn)含有LRR結(jié)構(gòu)域的受體蛋白編碼基因,這些基因在侵染階段表達模式不同。但是這些基因在卵菌中的功能尚無相關(guān)報道。為明確這些基因的功能,我們進行了以下研究:首先,我們從4個分類群體（植物、藻類、真菌、卵菌）中選擇14個具有代表性的物種,通過比較這些物種的完整基因組序列并進行仔細分析,發(fā)現(xiàn)卵菌的LRR-RLKs家族由111個成員組成。所有物種中的LRR-RLK蛋白質(zhì)序列理論上都形成一個典型的LRR-RLKs結(jié)構(gòu),包括LRRs、跨膜區(qū)、激酶域。在卵菌中,我... 

【文章來源】：南京農(nóng)業(yè)大學(xué)江蘇省 211工程院校 教育部直屬院校

【文章頁數(shù)】：178 頁

【學(xué)位級別】：博士

【文章目錄】：
ACKNOWLEDGEMENTS
摘要
ABSTRACT
CHAPTER ONE: INTRODUCTION
    1.1 Objectives
    1.2 Research Background
        1.2.1 Introduction to Phytophthora plant pathogen threatening agriculture and natural ecosystem
        1.2.2 Evolution
        1.2.3 Biology
        1.2.4 Genes for host-Phytophthora interaction
        1.2.5 Phytophthora capsici-a model oomycete species with broad host range
        1.2.6 Leucine-rich repeat receptor-like kinases(LRR-RLKs)
CHAPTER TWO: GENOME WIDE IDENTIFICATION OF LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASES (LRR-RLKs) IN OOMYCETES
    2.1 INTRODUCTION
    2.2 MATERIAL AND METHODS
        2.2.1 Identification and distribution of LRR-RLKs in oomycetes genome
        2.2.2 Preparation of P.capsici cultures and plants
        2.2.3 Collection of zoospore suspension and inoculation assay
        2.2.4 RNA extraction and real-time RT-PCR assay
    2.3 RESULTS
        2.3.1 Identification of LRR-RLKs in oomycetes
        2.3.2 Evolutionary analysis of LRR-RLKs in oomycetes
        2.3.3 Protein structure and conserved motif analysis of LRR-RLKs in oomycetes
        2.3.4 Differential expression profiles of P. capsici LRR-RLKs
    2.4 DISCUSSION
    2.5 CONCLUSION
CHAPTER THREE: PcLRR-RK1,AN LRR RECEPTOR KINASE REGULATESGROWTH, DEVELOPMENT AND PATHOGENESIS IN PHYTOPHTHORA CAPSICI
    3.1 INTRODUCTION
    3.2 MATERIAL AND METHODS
        3.2.1 Identification and distribution of LRR-RLKs in P. capsici genome
        3.2.2 Preparation of P. capsici cultures and plants
        3.2.3 Construction and transformation of recombinant plasmids
        3.2.4 RNA extraction and real-time RT-PCR assay
        3.2.5 Analysis of colony growth, zoosporangium development and production
        3.2.6 Inoculation assays on N. benthamiana and A. thaliana
        3.2.7 DAB staining and callose deposition assay
        3.2.8 Microscopic observation of cyst germination and pathogen establishment
    3.3 RESULTS
        3.3.1 Identification and distribution of LRR-RLKs in P. capsici genome
        3.3.2 PcLRR-RK1 is required for vegetative growth of P. capsici
        3.3.3 PcLRR-RK1 is essential for zoosporangium development and production
        3.3.4 Silencing of PcLRR-RK1 results in loss of zoospores infection abilities into hosttissues
        3.3.5 PcLRR-RK1 results into reduced germination of zoospores into host tissues
        3.3.6 Silencing of PcLRR-RK1 results in penetration reduction of P. capsici mycelium into the host tissues
        3.3.7 DAB staining and Callose deposition assay
    3.4 DISCUSSION
    3.5 CONCLUSION
CHAPTER FOUR: AN LRR RECEPTOR KINASE,PCLRR-RK2,IS CRUCIALLYREQUIRED FOR VEGETATIVE GROWTH, DEVELOPMENT AND PATHOGENESIS IN PHYTOPHTHORA CAPSICI
    4.1 INTRODUCTION
    4.2 MATERIAL AND METHODS
        4.2.1 Protein sequence selection and identification
        4.2.2 Maintenance of microbial strains, plants and culture conditions
        4.2.3 Plasmid construction and P. capsici transformation
        4.2.4 RNA isolation and quantitative real-time PCR
        4.2.5 Phenotypic characterization
        4.2.6 Pathogenecity assays
        4.2.7 DAB staining and Callose deposition assay
        4.2.8 Assays for the germination and penetration of zoospores into host tissues
    4.3 RESULTS
        4.3.1 Protein sequence selection and identification
        4.3.2 PcLRR-RK2 is required for vegetative development of P.capsici
        4.3.3 Silencing of PcLRR-RK2 interferes with zoosporangium development of P.capsici
        4.3.4 PcLRR-RK2 is important for zoospores infection abilities into host tissues
        4.3.5 PcLRR-RK2 results into reduced germination of zoospores into host tissues
        4.3.6 PcLRR-RK2 is required for penetration of P. capsici mycelium into host tissues
        4.3.7 PcLRR RK2 is important for host penetration,H_2O_2 accumulation and callose deposition
    4.4 DISCUSSION
    4.5 CONCLUSION
CHAPTER FIVE: PcLRR-RK3, AN LRR RECEPTOR KINASE IS REQUIRED FORVEGETATIVE DEVELOPMENT AND IN-PLANTA INFECTION PROCESSES IN PHYTOPHTHORA CAPSICI
    5.1 INTRODUCTION
    5.2 MATERIAL AND METHODS
        5.2.1 Protein sequence selection and identification
        5.2.2 Maintenance of plant and P.capsici cultures
        5.2.3 Plasmid Construction and transformation into P.capsici culture
        5.2.4 RNA extraction and qRT-PCR assay
        5.2.5 Phenotypic analysis of PcLRRK3-silenced transformants
        5.2.6 Pathogenecity assay on N. benthamiana
        5.2.7 Trypan blue staning,DAB staining and Callose deposition assay
        5.2.8 Observation of zoospores germination and infection into host tissues
        5.2.9 Agrobacterium-mediated transient gene expression in N. benthamiana
        5.2.10 Confocal Microscopy
    5.3 RESULTS
        5.3.1 PcLRR-RK3 is required for vegetative growth of P. capsici
        5.3.2 Impact of PcLRR-RK3 on the zoosporangium development and production
        5.3.3 PcLRR-RK3 is required for full virulence and in planta growth of P. capsici
        5.3.4 PcLRR-RK3 is essential for zoospores penetration and establishment into hostleaf tissues
        5.3.5 Sub-cellular localization of PcLRR-RK3
    5.4 DISCUSSION
    5.5 CONCLUSION
CHAPTER SIX: RNA SEQUENCING FOR COMPARATIVE TRANSCRIPTOMICANALYSIS OF PHYTOPHTHORA CAPSICI AFTER PcLRR-RLKs-SILENCING
    5.1 INTRODUCTION
    5.2 MATERIALS AND METHODS
        5.2.1 RNA extraction and qRT-PCR assay
        5.2.2 Library Preparation and Transcriptome analysis
    5.3 RESULTS
        5.3.1 Overview of the transcriptome
        5.3.2 Identification of differentially expressed genes(DEGs) in response to PcLRR-RLKs silencing
        5.3.3 GO annotation of DEGs
        5.3.4 PcLRR-RLKs regulate the growth of P. capsici
        5.3.5 PcLRR-RLKs regulate the infection process in P. capsici
        5.3.6 Validation of the transcriptome data by qRT-PCR
    5.4 DISCUSSION
    5.5 CONCLUSION
SUMMARY AND CONCLUSION
REFERENCES
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