Functional Characterization of miR828a Involved in the Negat

发布时间：2023-04-23 15:30

　　杨树是世界上种植面积最为广泛的经济林木之一,它不仅被广泛应用于经济、生态和环境保护等领域,而且作为模式树种应用于木本植物遗传转化和分子机制研究。杨树作为重要的速生树种,是木材的主要来源。木材的主要成分包括木质素(Lignin)、纤维素(Cellulose)和半纤维素(Hemicellulose),其中木质素是影响木材材性关键的成分之一。然而杨树中木质素生物合成机制尚不完全清楚。因此,阐明杨树中木质素合成的分子调控机制具有重要的意义。MicroRNA(miRNAs)是一种内源单链非编码小RNA,长度约为20-22个核苷酸,能够靶向调控目标基因的表达。大量研究表明,miRNAs在调控植物生长和发育过程中发挥着重要的作用。此外,miRNAs还能够调控细胞壁主要组分,如糖和木质素的生物合成。已有研究证实,miR828在柑橘粒化过程中调控木质素的生物合成,在红薯伤害处理下调控木质素和H₂O₂的积累。目前尚未报道miR828在杨树木材发育过程中是否参与木质素的生物合成调控。因此,本研究拟阐明miR828在杨树中木质素生物合成过程中的生物学功能。具体研究结...

【文章页数】：105 页

【学位级别】：博士

【文章目录】：
摘要
Abstract
Chapter1 Literature Review
    1.1 Function of wood
    1.2 Secondary cell wall
    1.3 Overview of the Lignin
    1.4 MYB transcription factor
    1.5 Biological functions of MYB transcription factor in plants
        1.5.1 Regulation of primary and secondary metabolism
        1.5.2 R2R3-MYB protein regulate plant development
        1.5.3 R2R3-MYB proteins respond to biotic and abiotic stresses
    1.6 MicroRNA
    1.7 MicroRNA biogenesis and processing
    1.8 Origin,conservation,and diversity of miRNAs in plants
    1.9 Biological function of plant microRNA
        1.9.1 MicroRNA in plant growth and development
        1.9.2 MicroRNA and stress response
    1.10 Identification of miRNA targets in plants
Chapter2 Introduction
    2.1 Background
    2.2 Problems and statements
    2.3 Technical route of this study
Chapter3 Cloning and Functional Analysis of miR828a in Poplar
    3.1 Introduction
    3.2 Materials and methods
        3.2.1 Plant materials
        3.2.2 Bacteria and plasmids
        3.2.3 Reagents,enzymes and kits
        3.2.4 Hormone and antibiotics
        3.2.5 Growth media for plants and bacteria
        3.2.6 Other reagents
        3.2.7 Plasmid extract reagents
        3.2.8 Equipment
        3.2.9 Bioinformatic analysis
        3.2.10 DNA extraction
        3.2.11 RNA extraction
        3.2.12 RNA quality detection
        3.2.13 RNA reverse transcription reaction
        3.2.14 miRNA specific reverse transcription reaction
        3.2.15 Quantitative Real-time PCR(qRT-PCR)
        3.2.16 Vector construction
        3.2.17 Enzyme digestion and ligation
        3.2.18 Preparation of chemically competent Escherichia coli(DH5α)
        3.2.19 Transformation of E.coli competent cells
        3.2.20 Confirmation of positive bacterial cloning
        3.2.21 Extraction of plasmid DNA
        3.2.22 Transformation of Agrobacterium tumefaciens
        3.2.23 Transformation of poplar leaves
        3.2.24 Identification of transgenic poplar
        3.2.25 GUS staining assay
        3.2.26 Observation of lignin components
        3.2.27 Lignin content determination
        3.2.28 Isolation of cell wall polysaccharide
        3.2.29 Prediction of miRNA target genes
    3.3 Results
        3.3.1 Isolation and characterization of miR828a in poplar
        3.3.2 Expression analysis of miR828a in poplar
        3.3.3 Construction and overexpression of miR828a
        3.3.4 Identification of the target genes of miR828a in poplar
    3.4 Discussion
Chapter4 Identification and Characterization of miR828a Target MYB
    4.1 Introduction
    4.2 Materials and methods
        4.2.1 Identification of poplar MYB gene
        4.2.2 Vector construction and plant transformation
        4.2.3 Degradome sequencing analysis
    4.3 Results
        4.3.1 MYB is a potential target gene of multiple miRNAs
        4.3.2 Expression analysis of miR828a target MYB genes
        4.3.3 Effect of MYB011 overexpression on secondary wall component in poplar
        4.3.4 MYB011 regulates gene encoding lignin Biosynthesis enzymes
    4.4 Discussion
Chapter5 Conclusion and Prospect
    5.1 Conclusion
    5.2 Prospect
References
Appendix
Acknowledgement
List of Published Papers



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