Cloning,Expression,and Functional Analysis of two Acetylchol

发布时间：2021-03-03 01:16

　　斜纹夜蛾（Spodoptera litura）是一种为害蔬菜和观赏性植物的世界性杂食性害虫。因斜纹夜蛾食性杂、具有长距离迁徙能力、世代多、繁殖能力强及易对农药产生抗性等特点,导致其防治困难。乙酰胆碱酯酶（AChE,EC3.1.1.7）在突触后膜催化水解递质乙酰胆碱（ACh）导致神经冲动终止,是有机磷酸酯（OP）和氨基甲酸酯（CB）两类杀虫剂的主要作用靶标。因此本研究克隆了2种斜纹夜蛾乙酰胆碱酯酶基因（SlAce1和SlAce2）,分析了这两个基因在斜纹夜蛾不同发育阶段和不同组织的表达水平;同时利用RNAi技术对SlAce1和SlAce2基因进行干扰,并对其分析生物学功能和表达模式进行分析。探究斜纹夜蛾Ace基因的功能为斜纹夜蛾的防治提供新思路。其主要研究结果如下:本文克隆了斜纹夜蛾2个乙酰胆碱酯酶基因（SlAce1和SlAce2）。序列分析表明:SlAce1 cDNA全长为2085bp,编码694个氨基酸残基;SlAce2 cDNA全长为1917bp,编码639个氨基酸残基。SlAce1和SlAce2都含有特定氨基酸—催化三联体、阴离子胆碱结合位点、氧离子空穴、酰基口袋、外周阴离子亚位... 

【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】：108 页

【学位级别】：博士

【文章目录】：
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


【参考文献】：
期刊论文
[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)



本文编号：3060315