DNA損傷是生物體無(wú)法避免的,因?yàn)樗梢杂杉?xì)胞內(nèi)源的反應(yīng)造成,也可以由外界環(huán)境中的各種脅迫造成。DNA損傷會(huì)威脅到基因組的完整性和細(xì)胞的活性。早期陸生植物小立碗蘚是植物干細(xì)胞再生和基因功能研究的一種模式植物。小立碗蘚在物理?yè)p傷信號(hào)的誘導(dǎo)下可以重編程分化的莖葉體葉片細(xì)胞再生出原絲體干細(xì)胞,且不依賴于外源添加的植物激素。在干細(xì)胞形成過(guò)程中,細(xì)胞基因組的完整性和細(xì)胞活性的保持非常關(guān)鍵�；谛×⑼胩\完善的再生研究體系,我們對(duì)DNA損傷在小立碗蘚干細(xì)胞再生過(guò)程中的作用及其背后的分子機(jī)制進(jìn)行了研究。主要研究結(jié)果如下:1.在小立碗蘚中,我們意外地發(fā)現(xiàn)短暫誘導(dǎo)DNA鏈斷裂可以誘導(dǎo)分化的葉片細(xì)胞重編程再生出原絲體頂端干細(xì)胞。經(jīng)過(guò)持續(xù)的培養(yǎng),這些DNA損傷誘導(dǎo)形成的原絲體干細(xì)胞可以正常的生長(zhǎng)并發(fā)育形成新的具有葉片的莖葉體。2.通過(guò)PI染色觀察,發(fā)現(xiàn)DNA鏈斷裂誘導(dǎo)的干細(xì)胞再生過(guò)程并不依賴于死細(xì)胞。3.彗星實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)在我們的實(shí)驗(yàn)條件下,DNA損傷試劑Zeocin和Camptocecin主要誘導(dǎo)DNA單鏈斷裂,DNA損傷試劑Bleomycin會(huì)同時(shí)誘導(dǎo)大量的DNA單鏈和雙鏈斷裂。4.在移除DNA損傷試劑后,... 

【文章來(lái)源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：115 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
ABSTRACT
摘要
Abbreviations
1 Introduction
    1.1 Stem cell and its formation
    1.2 Wounding-induced stem cell formation
    1.3 Stem cell formation in Physcomitrella
    1.4 Effects of DNA damage on stem cell formation
    1.5 Aims of this study
2 Materials and Methods
    2.1 Plant materials and growth conditions
    2.2 Analysis of cell death via fluorescence imaging
    2.3 Comet assay
    2.4 Accession numbers
    2.5 Plasmid construction
    2.6 Transformation and selection of transformants
    2.7 Removing the Zeocin resistance cassette
    2.8 Microscopy
    2.9 Transcriptome analysis
3 Results
    3.1 DNA strand break-inducing reagents enhance the reprogramming of excised leaf cells into chloronema stem cells
    3.2 DNA strand break-inducing reagents trigger the reprogramming of intact gametophore leaf cells into chloronema apical stem cells without wounding
    3.3 DNA strand break-induced stem cells have the developmental ability to produce gametophores
    3.4 DNA strand breaks are induced after Zeocin,CPT,or BLM treatment
    3.5 Stem cell formation rate positively correlates with the amount of SSBs induced by Zeocin within a certain range
    3.6 Few visible dead cells induced after cultivation with DNA strand break-inducing reagents
    3.7 Zeocin-induced reprogramming is independent of dead cells
    3.8 SSBs is repaired before Zeocin induced reprogramming
    3.9 STEMIN1 and CSP1 are involved in Zeocin-induced reprogramming
    3.10 STEMINs,but not CSPs,are necessary for DNA break-induced reprogramming
    3.11 STEMINs are not involved in repair of SSBs
    3.12 Transcriptome analysis during Zeocin induced reprogramming
    3.13 Transcript pattern of genes involved in wounding-induced reprogramming or DDR during and after the Zeocin treatment
    3.14 Generation of ATR and ATM deletion lines
    3.15 ATR,not ATM,is essential for DNA strand break-induced reprogramming
    3.16 ATR functions in the repair of SSBs induced by Zeocin
    3.17 STEMIN1 expression induced by DNA strand breaks depends on ATR
    3.18 Severe wounding may induce DNA strand breaks
4 Discussion
    4.1 DNA strand break-induced reprogramming is different from wounding-induced reprogramming
    4.2 DNA strand breaks that induce reprogramming is most likely SSBs
    4.3 Physiological significance of DNA strand break-induced reprogramming
    4.4 The gap between DNA strand breaks and stem cell formation
    4.5 Summary and prospect
References
Appendices
    Appendix1 Formula of BCDAT medium
    Appendix2 Comet assay protocol used in this study
    Appendix3 Supplemental videos
    Appendix4 Table of GO enrichment of DNA damage RNA-seq DEG clusters with p<0.05
Acknowledgements



本文編號(hào)：3204187