桔小实蝇精子发育相关基因和miRNA研究
发布时间：2021-10-20 07:29
橘小实蝇是一种毁灭性果蔬害虫,为害世界上热带和亚热带地区250多种水果和蔬菜。昆虫不育技术（SIT）是通过大量释放不育雄虫来降低昆虫的繁殖能力,从而控制昆虫种群数量、甚至根除害虫的一种害虫防治技术,被认为是控制采采蝇等害虫的有效手段。随着现代生物技术如转基因、RNAi、CRISR Cas9等进步和成熟,最近,基于遗传改造的不育昆虫技术已经成为现实。对精子发育中相关基因和mi RNAs的功能鉴定将为基于遗传改造的不育昆虫技术提供靶标。在本研究中,我们以橘小实蝇为研究对象,对精子发育中相关基因和mi RNAs进行了鉴定和分析其功能,筛选获得能有效降低雄虫繁殖能力的靶标基因TF gaga,orb2,tektin1和tssk1,并发现mi R-125-3p和mi R-276b-3p通过调控orb2基因的表达,影响精子发育,这些结果为建立不依赖辐射、基于遗传改造的害虫不育技术提供靶标基因和mi RNAs,并害虫防治提供新思路。主要研究结果如下:在本研究第一部分,我们选取八个和精子发育相关的基因TF gaga,parkin,orb2,netrin-A,netrin-B,tektin1,Ddx1和t...
【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】：107 页
【学位级别】：博士
【文章目录】：
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
本文编号：3446501
【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】：107 页
【学位级别】：博士
【文章目录】：
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
本文编号：3446501
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