橘小實(shí)蠅是一種毀滅性果蔬害蟲,為害世界上熱帶和亞熱帶地區(qū)250多種水果和蔬菜。昆蟲不育技術(shù)（SIT）是通過大量釋放不育雄蟲來降低昆蟲的繁殖能力,從而控制昆蟲種群數(shù)量、甚至根除害蟲的一種害蟲防治技術(shù),被認(rèn)為是控制采采蠅等害蟲的有效手段。隨著現(xiàn)代生物技術(shù)如轉(zhuǎn)基因、RNAi、CRISR Cas9等進(jìn)步和成熟,最近,基于遺傳改造的不育昆蟲技術(shù)已經(jīng)成為現(xiàn)實(shí)。對(duì)精子發(fā)育中相關(guān)基因和mi RNAs的功能鑒定將為基于遺傳改造的不育昆蟲技術(shù)提供靶標(biāo)。在本研究中,我們以橘小實(shí)蠅為研究對(duì)象,對(duì)精子發(fā)育中相關(guān)基因和mi RNAs進(jìn)行了鑒定和分析其功能,篩選獲得能有效降低雄蟲繁殖能力的靶標(biāo)基因TF gaga,orb2,tektin1和tssk1,并發(fā)現(xiàn)mi R-125-3p和mi R-276b-3p通過調(diào)控orb2基因的表達(dá),影響精子發(fā)育,這些結(jié)果為建立不依賴輻射、基于遺傳改造的害蟲不育技術(shù)提供靶標(biāo)基因和mi RNAs,并害蟲防治提供新思路。主要研究結(jié)果如下:在本研究第一部分,我們選取八個(gè)和精子發(fā)育相關(guān)的基因TF gaga,parkin,orb2,netrin-A,netrin-B,tektin1,Ddx1和t... 

【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(yè)數(shù)】：107 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
ABSTRACT
LIST OF ABRIVIATIONS
CHAPTER1:GENERAL INTRODUCTION AND LITERATURE REVIEW
    1.1.Background of oriental fruit fly
        1.1.1.Economic importance
    1.2.Management of B.dorsalis
        1.2.1.Cultural control
        1.2.2.Biological control
        1.2.3.Chemical control
        1.2.4.Genetic control
    1.3.Spermatogenesis in insects
        1.3.1.Genetic regulation of spermatogenesis
    1.4.RNA Interference(RNAi)
        1.4.1.RNAi mechanism
        1.4.2.RNAi-based pest controls
        1.4.3.Double stranded(dsRNA)uptake in insects
        1.4.4.Functional analysis through RNAi of insect’s genes related to spermatogensis
    1.5.MicroRNAs
        1.5.1.miRNA biogensis
        1.5.2.MicroRNAs regulators of spermatogenesis
    1.6.Research objectives
CHAPTER2:FUNCTIONAL CHARACTERIZATION OF GENES RELATED TO SPERMATOGENSIS OF BACTROCERA DORSALIS
    2.1.Introduction
    2.2.Materials and Methods
        2.2.1.Flies rearing
        2.2.2.Laboratory reagents
        2.2.3. Agarose gel preparation:
        2.2.4.RNA extraction
        2.2.5.First strand cDNA synthesis
        2.2.6.Primer design
        2.2.7.qRT-pCR for gene expression
        2.2.8.Sequence analysis and construction of phylogenetic tree
        2.2.9.Open reading frame PCR(ORF)cloning and sequencing
        2.2.10.Double-Stranded RNA(dsRNA)Synthesis
        2.2.11. Double-Stranded RNA (ds RNA ) synthesis
        2.2.12.Double-Stranded RNA(dsRNA)feeding
        2.2.13.Collection of sample
        2.2.14.Knock down qRT-PCR
        2.2.15.Reproductive capacity of male flies
        2.2.16.Spermatozoa counts
        2.2.17.Statistical analysis
    2.3.Results
        2.3.1.Selection of genes related to the spermatogenesis
        2.3.2.Expression of genes in different body tissues
        2.3.3.Sequence alignment& phylogenetic analysis of four selected genes of B.dorsalis
        2.3.4.RNAi Analysis
        2.3.5.Effect of RNAi of four genes on the reproductive capacity of males
        2.3.6.Effects of gene silencing on the quantity of spermatozoa
    2.4.Discussion
CHAPTER3:MIR-125-3P AND MIR-276B-3P REGULATE THE SPERMATOGENSIS OF BACTROCERA DORSALIS BY TARGETING ORB
    3.1.Introduction
    3.2.Materials and Methods
        3.2.1.Bioinformatics analysis
        3.2.2.Orb23′UTR cloning and ligation into expression vector
        3.2.3.Examining DNA by gel electrophoresis
        3.2.4.Bacterial transformation
        3.2.5.Plasmid Extraction and Purification
        3.2.6.miRNA mimics,agomirs and antagomiRs synthesis
        3.2.7.Quantitative real-time PCR
        3.2.8.Cell culture
        3.2.9.Dual-luciferase assay
        3.2.10.Dietary delivery of agomirs,antagomirs and dsRNAs to adult flies
        3.2.11. Reproductive capacity of male flies.
        3.2.12. Sperm viability assays and spermatozoa counts.
        3.2.13.Statistical analysis
    3.3.Results
        3.3.1.Target gene selection
        3.3.2. Prediction of mi RNAs targeting orb2
        3.3.3.Expression profiles of miRNAs targeting orb2 in different body tissues:
        3.3.4.Confirmation of orb2 as a common target of miR-125-3p and miR-276b-3p
        3.3.5.Effect of agomiR-125-3p and agomiR-276b-3p administration on expression of miR-125-3p and miR-276b-3p
        3.3.6. Effects of agomi R-125-3p and agomi R-276b-3p ingestion on m RNA of ob2
        3.3.7.Effects of agomirs administration on male fertility
        3.3.8.Investigation of number of spermatozoa and sperm viability
        3.3.9.RNAi of antagomiR-125-3p and antagomiR-276-3p treated individuals rescued the phenotype caused by dsorb2 treatment
    3.4.Discussion
CHAPTER4:SUMMARY
    4.1.Innovations
    4.2.Future perspectives
REFERENCES
Acknowledgements



本文編號(hào)：3446501