灰飞虱几种细胞色素P450单加氧酶对杀虫剂降解的催化能力分析
发布时间：2021-11-22 01:27
本文重点研究灰飞虱不同细胞色素P450单加氧酶（氧化酶）对杀虫剂的降解催化能力。灰飞虱是东南亚地区水稻的重要害虫,尤其是上世纪60年代以来,危害逐步加重,防控研究也广泛展开。灰飞虱是几种重要农作物病毒病的传播媒介,传播的病毒病甚至能使稻谷减产50%,严重威胁水稻生产和世界粮食安全。为此,生产上花费大量财力进行防控,以避免灰飞虱危害造成产量损失。在中国,上世纪60年代灰飞虱就通过传播水稻条纹叶枯病和黑条矮缩病等病毒病导致过严重减产,自此以后,灰飞虱一直被认为是重要害虫。近期研究发现,灰飞虱田间种群对不同杀虫剂已陆续产生了抗药性,而且目前已经日趋普遍,并进行了大量研究,现已弄清解毒能力增加是抗药性的重要机制,而P450氧化酶在杀虫剂解毒和抗药性形成中均发挥了重要作用。P450是不同生物体内广泛存在的亚铁血红色蛋白,所组成的P450氧化酶系可以利用不同化合物进行酶促反应,是生物体内具有多种生理功能的重要酶系。在昆虫体内,P450氧化酶可以降解杀虫剂,并促进多种内源化合物的转化。一般情况下,抗药性昆虫体内某些P450氧化酶基因会出现过量表达,由此认为这些P450参与了相关杀虫剂的解毒代谢,是抗...
【文章来源】：南京农业大学江苏省 211工程院校 教育部直属院校
【文章页数】：157 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]南昌地区灰飞虱的生活史、繁殖和越冬生物学特性[J]. 王柳风,傅淑,肖亮,陈超,薛芳森. 昆虫学报. 2013(12)
[2]灰飞虱对杀虫剂抗药性的研究进展[J]. 王彦华,吴长兴,赵学平,苍涛,陈丽萍,俞瑞鲜,吴声敢,王强. 植物保护. 2010(04)
[3]防治灰飞虱高效活性化合物和杀虫单剂及复配剂研究[J]. 刘宝生,王利华,郭慧芳,钟万芳,方继朝. 江苏农业学报. 2009(06)
[4]昆虫细胞色素P450基因的多样性、进化及表达调控[J]. 郭亭亭,姜辉,高希武. 昆虫学报. 2009(03)
[5]几类杀虫剂对灰飞虱的相对毒力及田间种群的抗药性现状[J]. 王利华,方继朝,刘宝生. 昆虫学报. 2008(09)
[6]浙江省灰飞虱对吡虫啉、锐劲特和毒死蜱的抗药性监测[J]. 张晓婕,陈建明,陈列忠,俞晓平. 浙江农业学报. 2007(06)
[7]灰飞虱的生态学特性及可持续控制途径的研究进展[J]. 李伟,郭慧芳,方继朝,王荣富. 安徽农业科学. 2007(08)
[8]灰飞虱种群暴发成灾原因剖析[J]. 刘向东,翟保平,刘慈明. 昆虫知识. 2006(02)
本文编号：3510655
【文章来源】：南京农业大学江苏省 211工程院校 教育部直属院校
【文章页数】：157 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]南昌地区灰飞虱的生活史、繁殖和越冬生物学特性[J]. 王柳风,傅淑,肖亮,陈超,薛芳森. 昆虫学报. 2013(12)
[2]灰飞虱对杀虫剂抗药性的研究进展[J]. 王彦华,吴长兴,赵学平,苍涛,陈丽萍,俞瑞鲜,吴声敢,王强. 植物保护. 2010(04)
[3]防治灰飞虱高效活性化合物和杀虫单剂及复配剂研究[J]. 刘宝生,王利华,郭慧芳,钟万芳,方继朝. 江苏农业学报. 2009(06)
[4]昆虫细胞色素P450基因的多样性、进化及表达调控[J]. 郭亭亭,姜辉,高希武. 昆虫学报. 2009(03)
[5]几类杀虫剂对灰飞虱的相对毒力及田间种群的抗药性现状[J]. 王利华,方继朝,刘宝生. 昆虫学报. 2008(09)
[6]浙江省灰飞虱对吡虫啉、锐劲特和毒死蜱的抗药性监测[J]. 张晓婕,陈建明,陈列忠,俞晓平. 浙江农业学报. 2007(06)
[7]灰飞虱的生态学特性及可持续控制途径的研究进展[J]. 李伟,郭慧芳,方继朝,王荣富. 安徽农业科学. 2007(08)
[8]灰飞虱种群暴发成灾原因剖析[J]. 刘向东,翟保平,刘慈明. 昆虫知识. 2006(02)
本文编号：3510655
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