DNA损伤诱导小立碗藓叶片细胞再编程形成干细胞的机理研究
发布时间：2021-05-24 12:22
DNA损伤是生物体无法避免的,因为它可以由细胞内源的反应造成,也可以由外界环境中的各种胁迫造成。DNA损伤会威胁到基因组的完整性和细胞的活性。早期陆生植物小立碗藓是植物干细胞再生和基因功能研究的一种模式植物。小立碗藓在物理损伤信号的诱导下可以重编程分化的茎叶体叶片细胞再生出原丝体干细胞,且不依赖于外源添加的植物激素。在干细胞形成过程中,细胞基因组的完整性和细胞活性的保持非常关键。基于小立碗藓完善的再生研究体系,我们对DNA损伤在小立碗藓干细胞再生过程中的作用及其背后的分子机制进行了研究。主要研究结果如下:1.在小立碗藓中,我们意外地发现短暂诱导DNA链断裂可以诱导分化的叶片细胞重编程再生出原丝体顶端干细胞。经过持续的培养,这些DNA损伤诱导形成的原丝体干细胞可以正常的生长并发育形成新的具有叶片的茎叶体。2.通过PI染色观察,发现DNA链断裂诱导的干细胞再生过程并不依赖于死细胞。3.彗星实验检测发现在我们的实验条件下,DNA损伤试剂Zeocin和Camptocecin主要诱导DNA单链断裂,DNA损伤试剂Bleomycin会同时诱导大量的DNA单链和双链断裂。4.在移除DNA损伤试剂后,...
【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】：115 页
【学位级别】：博士
【文章目录】：
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
本文编号：3204187
【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】：115 页
【学位级别】：博士
【文章目录】：
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
本文编号：3204187
本文链接：https://www.wllwen.com/projectlw/swxlw/3204187.html
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