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与鸭子黑色和白色羽毛色素沉着关联的microRNA的分析鉴定

发布时间:2022-01-20 07:48
  基因表达的转录后调控的一个主要机制是通过microRNA (miRNA)实现的。单个的miRNA可以在多种生物学过程中发挥关键作用,如细胞增殖和分化、凋亡和癌变。然而,由哪些特定miRNA可能调节羽毛色素沉着尤其是黑色素生成的机制仍然在很大程度上难以捉摸。因此,本研究旨在比较研究鸭黑色背羽和黑白色背羽二者黑色羽球的miRNA表达与鸭白色背羽和黑白色背羽二者白色羽球的miRNA表达,这些鸭分别是Cui Hei鸭、改鸭、纯系连城鸭和一个改鸭-连城鸭的F2群体。我们运用了Solexa测序、生物信息学和实验方法初步筛选了与鸭子羽毛色素沉着相关的miRNA。主要结果如下:(1)利用MiRDeep2软件鉴定了129个miRNA,其中121个与已知的鸡的miRNA相近,8个为新的miRNA。另外,使用DEGseq软件还在这两种组织中鉴定出了五个显著差异表达的miRNA,即miR-204-5p、miR-204-3p、miR-144-3p、 miR-2188-3p和miR-130a-5p。值得注意的是,miR-204最主要在黑色羽球中表达。(2)为了进一步验证测序数据,完成了10个序列已鉴定的miRNA... 

【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】:80 页

【学位级别】:硕士

【文章目录】:
ABSTRACT
中文摘要
ABBREVIATIONS AND ACRONYMS
1 INTRODUCTION
    1.1 Origin of the research question
    1.2 Melanogenesis
        1.2.1 Definition of Melanogenesis
        1.2.2 Melanin synthesis
    1.3 Research progress on candidate genes associated with black and white plumage pigmentation in ducks
    1.4 MicroRNA
        1.4.1 The Discovery of miRNA Regulation
        1.4.2 MiRNA biogenesis
        1.4.3 MiRNA profiling
        1.4.4 MiRNA identified in ducks
        1.4.5 MicroRNA and pigmentation
    1.5 Development and application of high throughput sequencing technology
        1.5.1 Next Generation Sequencing and miRNA Study
        1.5.2 Analysis tools for NGS data
        1.5.3 miRNA Target Prediction
    1.6 Aim of thesis
    1.7 Significance of the research
2 MATERIAL AND METHODS
    2.1 Materials
        2.1.1 Experimental animals
        2.1.2 Software and hardware
        2.1.3 Major reagents
    2.2 Methods
        2.2.1 Preparation of main reagents
        2.2.2 Sample collection,small library preparation
        2.2.3 Sequencing
        2.2.4 Data analysis
            2.2.4.1 Raw data processing and obtaining clean reads
            2.2.4.2 Identification of conserved and novel miRNAs
            2.2.4.3 Differential expression analysis
        2.2.5 Experimental validation of sequencing data by stem loop qPCR
        2.2.6 Tissue profiling of miR-204-5p
        2.2.7 MiRNA target predictions,pathway analysis and GO analysis
        2.2.8 Gene expression analysis and validation of miRNA and target gene interaction.
            2.2.8.1 qPCR
            2.2.8.2 Dual luciferase reporter assay
            2.2.8.3 Western blotting
3 RESULTS
    3.1 Overview of sequencing results
    3.2 Identification of Conserved and Novel miRNAs
    3.3 The first nucleotide bias of miRNA in duck feather bulbs
    3.4 Identification of miRNAs Isoform
    3.5 Identification of differentially expressed miRNAs
    3.6 Experimental validation of sequenced data
    3.7 Tissue expression pattern of miR-204
    3.8 MicroRNAs target predictions,KEGG pathway and GO analysis
    3.9 Expression analysis of miRNA target genes by qPCR
    3.10 Duck miR-27-3pand MITF gene interaction
        3.10.1 Dual luciferase reporter assay
        3.10.2 Western blotting
4 DISCUSSION
    4.1 MiRNAs identified in black and white feather bulbs library of ducks
    4.2 Identification of differentially expressed miRNAs between the feather bulbs
    4.3 MiRNA target prediction
5 SUMMARY
    5.1 The main findings of this study
    5.2 The innovative points of this research
    5.3 Limitations of the study
    5.4 Further research suggestions
CONCLUSION
REFERENCES
PUBLICATIONS
APPENDIX
ACKNOWLEDGEMENT


【参考文献】:
期刊论文
[1]Characterization of MicroRNA* Species in Peking Duck Skin[J]. ZHANG Li,XIE Xiu-juan,JIA Shan-gang,XIAO Mei,LIN Shu-dai,AN Li-long,LUO Wen,JIA Xin-zheng,NIE Qing-hua,ZHANG Xi-quan.  Journal of Integrative Agriculture. 2013(09)
[2]Identification of Novel and Differentially Expressed MicroRNAs in the Ovaries of Laying and Non-Laying Ducks[J]. YU De-bing,JIANG Bao-chun,GONG Jing,DONG Fu-lu,LU Ying-lin,YUE Hui-jie,WANG Zheng-chao,DU Wen-xing,GUO An-yuan.  Journal of Integrative Agriculture. 2013(01)



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