兩棲類無尾目在變態(tài)過程中會發(fā)生一系列復(fù)雜的形態(tài)學(xué)和生理學(xué)變化,以適應(yīng)其從水生幼體到陸生亞成體的生境轉(zhuǎn)變。這些變化主要表現(xiàn)為尾部的吸收,四肢的發(fā)育,鰓的退化及肺的發(fā)育,同時肝臟、骨骼和腸道等器官組織也會發(fā)生不同程度的重塑。為了研究兩棲動物在變態(tài)過程中腸道微生物菌群的變化特征;以及在骨骼發(fā)育早期過程中,軟骨內(nèi)骨化的特點(diǎn)及其分子機(jī)制。本試驗(yàn)以中華大蟾蜍幼體為研究對象,首先觀察和測量了中華大蟾蜍幼體在不同發(fā)育時期個體和其腸道的形態(tài)學(xué)變化;對變態(tài)前期(Gosner stage 30,G30)、臨近變態(tài)期(G38)、變態(tài)高峰期(G42)和變態(tài)完成期(G46)中華大蟾蜍腸道微生物菌群的16S rDNA V3-V4區(qū)域進(jìn)行測序,從而得出不同發(fā)育時期腸道微生物菌群的多樣性的分布特征。同時對中華大蟾蜍后肢骨、脊柱的軟骨和骨骼進(jìn)行組織學(xué)染色研究,觀察其在變態(tài)過程中的發(fā)育變化,并利用轉(zhuǎn)錄組測序和實(shí)時定量PCR技術(shù)研究不同發(fā)育時期(G30、G37、G42和G46)中華大蟾蜍軟骨內(nèi)骨化相關(guān)基因的表達(dá)特征,從而了解在變態(tài)過程中骨骼發(fā)育的分子機(jī)制。主要研究結(jié)果如下:1、形態(tài)學(xué)數(shù)據(jù)顯示:中華大蟾蜍蝌蚪及其腸道發(fā)育在G2...

【文章頁數(shù)】：90 頁

【學(xué)位級別】：碩士

【文章目錄】：
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
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