本文重點研究灰飛虱不同細胞色素P450單加氧酶（氧化酶）對殺蟲劑的降解催化能力�；绎w虱是東南亞地區(qū)水稻的重要害蟲,尤其是上世紀60年代以來,危害逐步加重,防控研究也廣泛展開�；绎w虱是幾種重要農(nóng)作物病毒病的傳播媒介,傳播的病毒病甚至能使稻谷減產(chǎn)50%,嚴重威脅水稻生產(chǎn)和世界糧食安全。為此,生產(chǎn)上花費大量財力進行防控,以避免灰飛虱危害造成產(chǎn)量損失。在中國,上世紀60年代灰飛虱就通過傳播水稻條紋葉枯病和黑條矮縮病等病毒病導致過嚴重減產(chǎn),自此以后,灰飛虱一直被認為是重要害蟲。近期研究發(fā)現(xiàn),灰飛虱田間種群對不同殺蟲劑已陸續(xù)產(chǎn)生了抗藥性,而且目前已經(jīng)日趨普遍,并進行了大量研究,現(xiàn)已弄清解毒能力增加是抗藥性的重要機制,而P450氧化酶在殺蟲劑解毒和抗藥性形成中均發(fā)揮了重要作用。P450是不同生物體內(nèi)廣泛存在的亞鐵血紅色蛋白,所組成的P450氧化酶系可以利用不同化合物進行酶促反應,是生物體內(nèi)具有多種生理功能的重要酶系。在昆蟲體內(nèi),P450氧化酶可以降解殺蟲劑,并促進多種內(nèi)源化合物的轉(zhuǎn)化。一般情況下,抗藥性昆蟲體內(nèi)某些P450氧化酶基因會出現(xiàn)過量表達,由此認為這些P450參與了相關(guān)殺蟲劑的解毒代謝,是抗... 

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

【文章頁數(shù)】：157 頁

【學位級別】：博士

【文章目錄】：
中文摘要
ABSTRACT
CHAPTER ONE GENERAL INTRODUCTION AND LITERATURE OF REVIEW
    1. INTRODUCTION
        1.1 Background of the present study
        1.2 Rationale/justification of the present study
        1.3 Scope of the present study
        1.4 Objectives of the present study
    2. LITERATURE OF REVIEW
        2.1 Small brown planthopper
        2.2 Insecticides
        2.3 Cytochrome P450 monooxygenases (P450s)
        2.4 Cytochrome P450 monooxygenases (P450s) in insects
        2.5 Cytochrome P450s and redox partners
        2.6 Insecticides degradation mechanism by insect P450s
CHAPTER TWO MATERIALS AND METHODS
    2.1 Chemicals and Reagents
    2.2 Biological Materials
        2.2.1 Experimental insects
        2.2.2 Spodoptera frugiperda (Sf9) cell lines
    2.3 Tested P450 genes (Family 4) of L. striatellus
    2.4 Functional expression of P450 genes in Sf9 cell and microsomal protein isolation
        2.4.1 Gene Cloning
        2.4.2 Construction of expression vector (recombinant plasmid)
        2.4.3 Cell culture and stable transfer
        2.4.4 Preparation of cell lysate/microsome
    2.5 Total RNA Isolation, cDNA Synthesis and RT-PCR detection of expression in Sf9cell
    2.6 SDS-PAGE analysis
    2.7 Carbon monoxide (CO) difference spectroscopy
    2.8 Determination of Enzyme activities
        2.8.1 Oxidative metabolism against model substrate
        2.8.2 Metabolism of probe substrates
    2.9 HPLC analysis of insecticide degradation
    2.10 UPLC-MS and MS/MS analysis and identification of metabolites
    2.11 P450 homology modeling and substrate docking
CHAPTER THREE DELTAMETHRIN IS DEGRADED BY CYP439A1v3; A CYTOCHROME P450OVER-EXPRESSED IN RESISTANT STRAIN OF Laodelphax striatellus
    3.1 Abstract
    3.2 Introduction
    3.3 Materials and Methodology
        3.3.1 Chemical and biological materials
        3.3.2 P450 gene used in this study
        3.3.3 Vector construction for functional Expression in Sf9 cell
        3.3.4 CYP439A1v3 transfection in Sf9 Cells
        3.3.5 Methodology for biochemical analysis of P450 gene
    3.4 Results
        3.4.1 Functional expression of L. striatellus CYP439Alv3
        3.4.2 CYP439A1v3 catalytic activity against standard P450 model substrates
        3.4.3 CYP439A1v3 catalytic activity for insecticide metabolism
        3.4.4 Identification of deltamethrin metabolites
        3.4.5 CYP439A1v3 homology modeling and substrate docking
    3.5 Discussion
CHAPTER FOUR RESISTANCE IRRELEVANT CYP417A2v2 WAS FOUND DEGRADINGINSECTICIDE IN Laodelphax striatellus
    4.1 Abstract
    4.2 Introduction
    4.3 Materials and Methodology
        4.3.1 Chemical and biological materials
        4.3.2 P450 genes used in this study
        4.3.3 Vector construction for functional Expression in Sf9 cell
        4.3.4 P450 genes transfection in Sf9 Cells
        4.3.5 Methodology for biochemical analysis of P450 gene
    4.4 Results
        4.4.1 Functional expression of L. striatellus P450s
        4.4.2 Enzymatic activity of CYP417A2v2 with CO-difference spectrum
        4.4.3 CYP417A2v2 catalytic activity against standard P450 model substrates
        4.4.4 CYP417A2v2 capability to metabolize insecticides
        4.4.5 Identification of imidacloprid metabolite
    4.5 Discussion
CHAPTER FIVE FIPRONIL WAS FOUND TO BE DEGRADED BY A CYTOCHROME P450MONOOXYGENASE CYP426A1 IN Laodelphax striatellus
    5.1 Abstract
    5.2 Introduction
    5.3 Materials and Methodology
        5.3.1 Chemical and biological materials
        5.3.2 P450 gene used in this study
        5.3.3 Vector construction for functional Expression in Sf9 cell
        5.3.4 CYP426A1 transfection in Sf9 Cells
        5.3.5 Methodology for biochemical analysis of P450 gene
    5.4 Results
        5.4.1 Functional expression of L. striatellus CYP426A1
        5.4.2 Catalytic activity of CYP426Alagainst standard P450 model substrates
        5.4.3 CYP426A1 catalytic activity for insecticide metabolism
    5.5 Discussion
SUMMARY
REFERENCES
PUBLICATIONS
ACKNOWLEDGEMENTS


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