小地老虎Agrotis ipsilon是一種破壞力很強的遷飛性鱗翅目害蟲,危害世界范圍內的多種作物。性信息素合成激活肽受體（PBANR）,調節(jié)多種鱗翅目昆蟲性信息素的合成。能夠與調控性信息素合成中扮演重要角色的GPCRs結合。在鱗翅目成蟲中,交配能夠引起雌蟲短時間終止性信息素的產生。本文通過RACE-PCR方法,從小地老虎雌蟲性腺中克隆獲得小地老虎PBANR全長基因,命名為Aips-PBANR,該基因由3539個堿基組成。蛋白序列比對表明,Aips-PBANR與鱗翅目其他昆蟲的PBANR具有較高的序列一致性。利用RT-PCR方法,從小地老虎雌蟲性腺和雄蟲生殖器中均檢測到Aips-PBANR基因的表達。qRT-PCR該基因在成蟲不同組織和日齡的表達特征進行研究。結果表明,Aips-PBANR在雌蟲性腺中的表達量最高,在雄蟲的生殖器中也檢測到較低的表達;羽化后第四天上午2:00基因表達量最高;另外,qRT-PCR結果表明,交配能夠明顯抑制Aips-PBANR基因的表達,因此進一步證明交配能夠抑制昆蟲性信息素的產生。RNA干擾技術（RNAi）作為害蟲防治的新方法具有很大的應用潛力。然而,在不... 

【文章來源】：中國農業(yè)科學院北京市

【文章頁數】：138 頁

【學位級別】：博士

【文章目錄】：
中文摘要
Abstract
Abbreviations
CHAPTER 1 Introduction and review of literature
    1.1. Black Cutworm
    1.2. Distribution of Black cutworm
    1.3. Life History and Developmental Stages of BCW
        1.3.1. Life History of BCW
        1.3.2. Egg Stage
        1.3.3. Larval Stage
        1.3.4. Pupal Stage
        1.3.5. Adult Stage
    1.4. Factors and Damages by BCW
    1.5. Significance and Migration of BCW
    1.6. Economic Importance
    1.7. Management of BCW
        1.7.1. Survey and Trapping
        1.7.2. Chemical Control
        1.7.3. Cultural and Mechanical Control
    1.8. Molecular Techniques used the present study
        1.8.1. Rapid Amplification of cDNA Ends (RACE)
        1.8.2. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
        1.8.3. Quantitative Real-Time PCR (qRT-PCR)
        1.8.4. Basic Principals of Real Time PCR
        1.8.5. Chemistries of Real Time PCR
        1.8.6. DNA Binding Dye Chemistry
        1.8.7. Probe Based Chemistry
        1.8.8. Primer and Probe designing for qRT-PCR
        1.8.9. The qRT-PCR Reaction Performance
        1.8.10. PCR Reaction Efficiency
        1.8.11. PCR Reaction Precision
        1.8.12. PCR Reaction Sensitivity
    1.9. Objectives
CHAPTER 2 Molecular Cloning, differential expression and mating interruption of Pheromone Biosynthesis Activating Neuropeptide Receptor, a sex pheromone gene in blackcutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera:Noctuidae) adults
    2.1. Introduction
    2.2. Materials and methods
        2.2.1. Ethics Statement
        2.2.2. Insect Material and Rearing Method
        2.2.3. Pheromone Gland Dissection Method
        2.2.4. Total RNA Extraction
        2.2.5. Synthesis of cDNA
        2.2.6. Tissue distribution, different time rythms and mating effect studies
        2.2.7. Molecular Cloning and sequencing of A. ipsilon PBANR
        2.2.8. Full length PBANR acquired by Rapid amplification of cDNA ends(RACE)
        2.2.9. Basic Bioinformatic Analysis
        2.2.10. Aips-PBANR Expression by Reverse transcription PCR (RT-PCR)
        2.2.11. Expression of Aips-PBANR by Quantitative Real-Time PCR
        2.2.12. Statistical Analysis
    2.3. Results
        2.3.1. Cloning and Sequencing of Aips-PBANR
        2.3.2. Phylogenetic Relationship of Aips-PBANR
        2.3.3. RT-PCR analysis of Aips-PBANR expression
        2.3.4. Quantitative RT-PCR (qRT-PCR) Analysis of Aips-PBANR
    2.4. DISCUSSION
CHAPTER 3 Knockdown of pheromone biosynthesis-activating neuropeptide receptor (PBANR) in black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera:Noctuidae) adults by RNAinterference
    3.1. Introduction
        3.1.1. Sex Pheromone
        3.1.2. RNA interference (RNAi)
        3.1.3. Need for RNAi
        3.1.4. RNA interference by siRNA
        3.1.5. RNA interference by dsRNA
        3.1.6. Different dsRNA Uptake methods used in RNAi
        3.1.7. Advantages and disadvantages of RNAi by dsRNA
        3.1.8. Important factors influencing RNAi through dsRNA
            3.1.8.1 Concentration of dsRNA
            3.1.8.2 Nucleotide sequence
            3.1.8.3 Length of dsRNA fragment
            3.1.8.4 Persistence of the silencing effect
            3.1.8.5 Life stage of the target organism
    3.2. Materials and methods
        3.2.1. Insect Culture and Rearing Methods
        3.2.2. Tissue Collection
        3.2.3. RNA extraction and cDNA synthesis
        3.2.4. Molecular cloning
        3.2.5. Synthesis and Injection of siRNA and dsRNA
        3.2.6. Quantitative Real-Time PCR (qRT-PCR) analysis
        3.2.7. Data Analysis
    3.3. Results
        3.3.1. Cloning and Sequencing of Aips-PBANR
        3.3.2. Quantitative RT-PCR (q RT-PCR) Analysis of Aips-PBANR expression
        3.3.3. Effect of si RNA treatment on Aips-PBANR transcript level
        3.3.4. Effect of dsRNA treatment on Aips-PBANR transcript level
    3.4. Discussion
CHAPTER 4 The influence of different temperatures on mating traits, oviposition andlogivity of black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera:Noctuidae)
    4.1. Introduction
    4.2. Materials and methods
        4.2.1. Study Organism
        4.2.2. Insect Colony Rearing
        4.2.3. Temperature and Mating Conditions
        4.2.4. Mating Observations
        4.2.5. Oviposition, Fecundity and Adult Longivity
        4.2.6. Statistical Data analysis
    4.3. Results
        4.3.1. Mating Activity
        4.3.2. Oviposition Traits
        4.3.3. Adult Longivity
    4.4. Discussion
CHAPTER 5 Conclusions and Recommendations
References
Acknowledgements
Author biography


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