中华大蟾蜍早期骨化的发生机制研究及变态过程中肠道微生物菌群多样性的研究

发布时间：2023-09-18 18:44

　　两栖类无尾目在变态过程中会发生一系列复杂的形态学和生理学变化,以适应其从水生幼体到陆生亚成体的生境转变。这些变化主要表现为尾部的吸收,四肢的发育,鳃的退化及肺的发育,同时肝脏、骨骼和肠道等器官组织也会发生不同程度的重塑。为了研究两栖动物在变态过程中肠道微生物菌群的变化特征;以及在骨骼发育早期过程中,软骨内骨化的特点及其分子机制。本试验以中华大蟾蜍幼体为研究对象,首先观察和测量了中华大蟾蜍幼体在不同发育时期个体和其肠道的形态学变化;对变态前期(Gosner stage 30,G30)、临近变态期(G38)、变态高峰期(G42)和变态完成期(G46)中华大蟾蜍肠道微生物菌群的16S rDNA V3-V4区域进行测序,从而得出不同发育时期肠道微生物菌群的多样性的分布特征。同时对中华大蟾蜍后肢骨、脊柱的软骨和骨骼进行组织学染色研究,观察其在变态过程中的发育变化,并利用转录组测序和实时定量PCR技术研究不同发育时期(G30、G37、G42和G46)中华大蟾蜍软骨内骨化相关基因的表达特征,从而了解在变态过程中骨骼发育的分子机制。主要研究结果如下:1、形态学数据显示:中华大蟾蜍蝌蚪及其肠道发育在G2...

【文章页数】：90 页

【学位级别】：硕士

【文章目录】：
摘要
Abstract
Chapter 1 Review
    1.1 Amphibian metamorphosis
        1.1.1 The overview of amphibian metamorphosis
        1.1.2 Major changes in anuran body during metamorphosis
        1.1.3 Remodeling of intestine in amphibian through metamorphosis
        1.1.4 Remodeling of skeleton in amphibian through metamorphosis
    1.2 16S rDNA sequencing for bacterial identification in amphibian
        1.2.1 Amphibian gut microbiota
        1.2.2 16S ribosomal RNA gene sequencing technology
        1.2.3 Application of 16S rDNA sequences in microbial ecology
    1.3 Transcriptome sequencing analysis of the endochondral ossification mechanismsof amphibian
        1.3.1 Endochondral ossification
        1.3.2 Transcriptome sequencing
        1.3.3 Application of transcriptome sequencing analysis in endocho -ndralossification mechanisms of amphibian
Chapter 2 Changes in intestinal microbiota of Bufo gargarizans duringmetamorphosis
    2.1 Introduction
    2.2 Materials and methods
        2.2.1 Study animal and sample collection
        2.2.2 Morphological observations and measurements
        2.2.3 DNA-extraction and PCR amplification
        2.2.4 Illumina MiSeq sequencing
        2.2.5 Processing of sequencing data
        2.2.6 Statistical analyses
    2.3 Results
        2.3.1 Tadpole and its intestine morphology
        2.3.2 Morphological measurement
        2.3.3 Taxonomic diversity
        2.3.4 OTUs distribution across different developmental stages
        2.3.5 Composition of the intestinal microbiota at various taxonomic levels
        2.3.6 Comparison of dominant bacterial communities between aquatic andterrestrial stages
        2.3.7 Effects of morphological changes on intestinal microbiota
    2.4 Discussion
Chapter 3 Transcriptome analyses reveal molecular mechanisms mediatingendochondral ossification in Bufo gargarizans through metamorphosis
    3.1 Introduction
    3.2 Methods
        3.2.1 Specimen collection
        3.2.2 Skeletal development
        3.2.3 RNA extraction and cDNA library construction
        3.2.4 Transcriptome sequencing, de novo assembly and functional annotation
        3.2.5 Differential gene expression analysis
        3.2.6 Real-time quantitative PCR
    3.3 Results
        3.3.1 Morphological data
        3.3.2 Transcriptome assembly and annotation
        3.3.3 Differential expressed genes in B.gargarizans during metamor phosis
        3.3.4 Functional annotation of differentially expressed unigene set
        3.3.5 The expression profiles of genes related to the initiation of skeletaldevelopment
        3.3.6 Real-time quantitative PCR
    3.4 Discussion
Chapter 4 Conclusion
References
致谢



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