兰州大学机构库 >生命科学学院
cytomixis活体研究体系的建立以及永双光子显微镜对烟草花药细胞中cytomixis的初步活体观察
Alternative TitleEstablishment of in vivo Observation System of Cytomixis and Its Primary Study in Intact Living Anthers of Tobacco with Two-photon Microscopy
刘恒
Thesis Advisor郑国锠
2002-05-12
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keywordcytomixis 烟草花药 Histone2B CFP 激光共聚焦荧
Abstract发现cytomixis现象已一个世纪,但是对于该现象的真实性目前仍然存在较大的争议,而最有说服力的便是对cytomixis现象进行活体观察。本文的目的即在于建立cytomixis的活体研究体系并对该现象进行活体观察。 本文首先构建了染色质的特异组成成分Histone2B(H2B)与CFP的融合蛋白植物表达载体,并将H2B-CFP置于CaMV35S启动子的控制之下,以烟草为材料,通过叶盘法成功的将H2B-CFP导入烟草, PCR和Southern blotting都证明在转基因烟草中含有目的片断。利用荧光显微镜,在转基因烟草多种组织的细胞核中都观察到了CFP的绿色荧光。在激光共聚焦显微镜下做了转基因烟草根尖细胞中PI和H2B-CFP两种荧光的共定位,证明了H2B-CFP荧光确实是细胞核发出的荧光。用流式细胞仪对转基因和未转基因烟草细胞周期进行了分析,结果表明H2B-CFP融合蛋白的表达对植物的细胞周期没有影响。用激光共聚焦荧光显微镜进一步对获得的转基因烟草的根尖细胞进行了活体动态观察,观察到了根尖细胞有丝分裂后期染色质分离的连续动态过程。由此成功建立了cytomixis的活体研究体系。 用激光共聚焦荧光显微镜对表达H2B-CFP的烟草花药表皮细胞进行了活体动态观察,发现细胞核的形状经常发生变化,周围常常伸出一些小的突起,形成染色质小球,这些小球处于活跃运动状态,可以观察到从一个细胞的核中伸出部分染色质迅速转移到另一个细胞的核中的现象。首先从一个核中伸出一个长丝状的突起,随后丝状突起与原来的细胞核断裂,与原来核相连的染色质丝缩回到原来的细胞核中,而断裂脱离后的部分染色质向相邻细胞核移动,并最终和该细胞核融合在一起。用双光子显微镜对表达H2B-CFP的烟草花药更深层次的细胞核的动态观察中,也观察到了正在进行着穿壁运动的细胞核的动态过程, 穿壁的过程与在电镜和光镜中观察到的结果一致。一个细胞核中的染色质从胞间通道穿过细胞壁分别到达相邻的两个细胞中并形成染色质小球,然后穿过的染色质越来越多,形成的染色质小球也逐渐增大,最后穿壁转移到相邻的一个细胞中的染色质形成一个新的细胞核,同时还发现穿壁后形成的细胞核离开原有位置,向细胞另一侧移动的现象,这种移动表现为一种整体的细胞核移动。本研究直接证明了穿壁现象的真实性。同时在烟草花药表皮和中层细胞中,穿壁的发生是不同步的,只能看到个别细胞的cytomixis现象。 本研究中,压片和对花药壁细胞的酶解都会使得细胞的活动减弱,细胞核的运动停滞。说明外界的影响只会降低cytomixis的发生,是一种促退,而不是促进染色质的穿壁运动。与本实验室多年的研究结果相符。
Other AbstractAs an old phenomenon described one century ago, the debate of the authenticity of cytomixis has still existed now. Observation in vivo is the most convincing approach to resolve the problem, which is the goal of this study. The Histone2B (H2B) and CFP fusion protein has been constructed and controlled by CaMV35S promotor. With leaf disc method, the H2B-CFP has been transferred to tobacco and confirmed by PCR and Southern hybridization. The CFP fluorescence in the nuclei in many tissues of transgenic tobacco has been observed with fluorescent microscope. The PI and H2B-CFP fluorescence co localization in the nuclei of root tips of transgenic tobacco, indicating that the H2B-CFP fluorescence was indeed localized in the nuclei. Fluorescence-activated cell sorting (FACS) analysis demonstrated that the regulation of the cell cycle is not affected by the expression of H2B-CFP. Further dynamic observation of root tips of transgenic plants with laser scanning confocal microscope (LSCM) showed the real dynamic process of the separation of chromosome during the anaphase of mitosis. These results indicated that the living observation system of cytomixis has been established successfully. Under LSCM, in epidermal cells of tobacco anther expressing H2B-CFP, the nuclei changed their shape frequently. Some of small protuberance often extruded out of the periphery of nucleus and formed small chromatin balls, and these dynamic small balls could migrate around the nucleus. More important phenomenon is the migration of part of chromatin from one of nucleus in a cell to another. Firstly, a long threadlike protuberance extruded out of one nucleus, then the threadlike protuberance ruptured from the primary nucleus, one part remained continuous with the original nucleus retracted back, but the other separated part changed its shape into sphere and moved to the nucleus lying in another cell and finally fused with it. Cytomixis in the more deeper cells below epidermal cells was also observed under two-photon microscope, which was consistent with that observed in light and electron microscope previously. The chromatin of one nucleus migrated through the cytoplasmic channel to the two adjacent cells and formed chromatin balls, then more and more chromatins passed through with larger and larger chromatin balls formed, finally these CBs fused to form a new nucleus in one of the two adjacent cells. The whole new-formed nucleus also migrated to the other side of the adjacent c...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222018
Collection生命科学学院
Recommended Citation
GB/T 7714
刘恒. cytomixis活体研究体系的建立以及永双光子显微镜对烟草花药细胞中cytomixis的初步活体观察[D]. 兰州. 兰州大学,2002.
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