發(fā)布時間：2021-03-03 01:16

　　斜紋夜蛾（Spodoptera litura）是一種為害蔬菜和觀賞性植物的世界性雜食性害蟲。因斜紋夜蛾食性雜、具有長距離遷徙能力、世代多、繁殖能力強(qiáng)及易對農(nóng)藥產(chǎn)生抗性等特點,導(dǎo)致其防治困難。乙酰膽堿酯酶（AChE,EC3.1.1.7）在突觸后膜催化水解遞質(zhì)乙酰膽堿（ACh）導(dǎo)致神經(jīng)沖動終止,是有機(jī)磷酸酯（OP）和氨基甲酸酯（CB）兩類殺蟲劑的主要作用靶標(biāo)。因此本研究克隆了2種斜紋夜蛾乙酰膽堿酯酶基因（SlAce1和SlAce2）,分析了這兩個基因在斜紋夜蛾不同發(fā)育階段和不同組織的表達(dá)水平;同時利用RNAi技術(shù)對SlAce1和SlAce2基因進(jìn)行干擾,并對其分析生物學(xué)功能和表達(dá)模式進(jìn)行分析。探究斜紋夜蛾Ace基因的功能為斜紋夜蛾的防治提供新思路。其主要研究結(jié)果如下:本文克隆了斜紋夜蛾2個乙酰膽堿酯酶基因（SlAce1和SlAce2）。序列分析表明:SlAce1 cDNA全長為2085bp,編碼694個氨基酸殘基;SlAce2 cDNA全長為1917bp,編碼639個氨基酸殘基。SlAce1和SlAce2都含有特定氨基酸—催化三聯(lián)體、陰離子膽堿結(jié)合位點、氧離子空穴、�；诖⑼庵荜庪x子亞位... 

【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁數(shù)】：108 頁

【學(xué)位級別】：博士

【文章目錄】：
Abstract
摘要
Chapter 1. LITERATURE REVIEW
    1.1 Introduction
        1.1.1 Lepidopteran insect pests
        1.1.2 Common cutworm, Spodoptera litura Fabricius: Insect Pest
        1.1.3 Common Name
        1.1.4 Classification
        1.1.5 Economic importance
        1.1.6 Morphology
        1.1.7 Life cycle
        1.1.8 Insecticide Resistance in Spodoptera litura
        1.1.9 Plant-Insect Interactions
    1.2 Acetylcholinesterase
        1.2.1 Distribution of Acetylcholinesterase
        1.2.2 Insecticide Target Sites and Structural Biology of Acetylcholinesterase
        1.2.3 Acetylcholinesterase insecticide Resistance Mechanisms
        1.2.4 Gene encoding acetylcholinesterase in insects
    1.3 RNA interference
        1.3.1 RNA interference
        1.3.2 A short history of the innovation of RNAi
        1.3.3 RNAi Pathways and Mechanism
        1.3.4 Use of RNAi
    1.4 RNAi in insects
        1.4.1 Preliminary RNAi trials in insects
        1.4.2 Study on the function of insect genes using RNAi methods
        1.4.3 Techniques of dsRNA transfer to Insects
        1.4.4 Use of RNAi in insect pest control strategies
        1.4.5 Susceptibility to RNAi
    1.5 Objectives of the study
Chapter 2. Cloning and expression of two Ace genes in Spodoptera litura
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Laboratory Equipment:
        2.2.2 Reagents and Expression vectors
        2.2.3 Other Reagents
    2.3 Methods
        2.3.1 Cloning of cDNA fragments of two SlAce genes
        2.3.2 Insects
        2.3.3 RNA extraction
        2.3.4 First Strand cDNA Synthesis
        2.3.5 Polymerase Chain Reaction （PCR）
        2.3.6 Purification of DNA using DNA Gel Extraction Kit
        2.3.7 DNA ligation with pGEM-T Easy Vector
        2.3.8 Transformation of plasmid DNA into Bacteria host （E. coli）
        2.3.9 Bacteria PCR
        2.3.10 Phylogenetic analysis
        2.3.11 SlAce genes expression pattern in different developmental stages and tissues
        2.3.12 Statistical analysis
    2.4 Results
        2.4.1 Sequence analysis of Spodoptera litura AChE genes
        2.4.2 SlAce genes expression profiles in S. litura
    2.5 Discussion
    2.6 Conclusion
Chapter 3. Functional analysis of two Ace genes in Spdoptera litura
    3.1 Introduction
    3.2 Methods
        3.2.1 dsRNA synthesis and application
        3.2.2 SlAce knockdown determination using qRT-PCR
        3.2.3 Statistical analysis
    3.3 Results
        3.3.1 Gene silencing through larvae injection of dsSlAce
        3.3.2 Injection with dsRNA delays pupation and emergence
        3.3.3 dsAce2 injection affects egg laying, egg hatching and progeny development
    3.4 Discussion
References
Appendix I
Appendix II
Scientific Publications
Acknowledgements


【參考文獻(xiàn)】：
期刊論文
[1]Ace2,rather than ace1,is the major acetylcholinesterase in the silkworm,Bombyx mori[J]. Hui-Juan Chen, Zhen Liao, Xiao-Ming Hui, Guo-Qing Li, Fei Li and Zhao-Jun HanDepartment of Entomology, Nanjing Agricultural University, Nanjing, China.  Entomologia Sinica. 2009 (04)



本文編號：3060315