【作者】 杨远媛；

【导师】 郑彩霞；

【作者基本信息】 北京林业大学 ， 植物学， 2008， 博士

【摘要】 辽宁兴城油松种子园的油松(Pinus tabulaeformis Carr.)雌性不育株(28号无性系)表现为球果能正常长大,但雌配子体由于游离核有丝分裂不能继续进行而停止发育,导致胚珠败育。该不育系具有稳定的遗传特性,是研究木本植物雌配子体发育调控机制的极好材料。为进一步分析该不育系游离核停止分裂的原因,本论文在系统研究油松28号不育系游离核停止分裂前后细胞形态学和组织化学的基础上,以油松种胚为材料建立了油松悬浮细胞系,采用细胞同步化技术、流式细胞仪技术、蛋白质双向电泳技术等研究手段初步分析了油松细胞周期调控机制;并进一步利用免疫印迹、免疫组化技术探讨了细胞周期相关调控因子在游离核分裂关键时期的表达。研究结果为揭示裸子植物雌配子体发育过程中细胞周期调控因子的表达与调控机制提供了非常有价值的信息,对拓展木本植物发育生物学的研究领域,以及揭示植物雌性不育机制等都具有十分重要的意义。1.利用油松雌配子体分离、电镜观察、细胞组织化学等技术,观察比较了在游离核停止分裂前后油松11号可育系和28号不育系珠被、珠心、雌配子体及游离核的显微结构、超微结构、营养物质的动态变化。发现油松不育系雌配子体发育停止在游离核时期,该时期不育系游离核等细胞器出现异常,细胞核膨大,核仁消失,核内染色质发生汇聚、凝集,在雌配子体细胞质中含有大量的异常线粒体,其形态异常膨大或延长,内部结构模糊不清,而小液泡的数量明显减少,甚至看不到较大的液泡,并且有部分液泡膜出现凹陷或裂解。另外不育系胚珠珠心组织细胞异常加厚,多糖物质出现异常积累。2.油松悬浮细胞细胞周期的研究结果显示,处于指数生长的油松悬浮细胞用3mM HU处理24h后,细胞恢复生长3h时,90.71%的细胞被阻断于G1期,恢复生长27h时,M/A分裂指数达到最大值,并且有40%的细胞进入G2期。分析同步化处理后恢复生长3h和27h的细胞蛋白质组,发现G1期细胞特有蛋白质点11个,M期细胞新增加4个。与G1期相比,M期表达量明显上调的蛋白质点7个,明显下调的蛋白质点3个。差异蛋白质主要分布在18kDa/pI6.3-30kDa/pI7.5和60kDa/pI6.5-70kDa/pI7.2两个区域。推测这些蛋白质可能参与了不同时期细胞周期的调控过程。3. CaM和类Cyclin C蛋白在雌配子体游离核有丝分裂过程中起着重要作用。CaM,类Cyclin C蛋白在油松细胞周期过程中呈现出相似的周期性变化,即G1期含量较少,S期大量积累,到G2期CaM含量有所减少,在下一个细胞周期S期又增加,表明这些因子与油松细胞周期调控有关。CaM和类Cyclin C蛋白在油松胚珠中的分布具有时空调控特征。在游离核旺盛分裂期,珠被大细胞层中CaM的分布明显减少,而外侧珠被组织中CaM的分布明显增加,推测珠被组织可能作为产生或集散CaM的部位,通过钙(Ca2+)信号系统调控了游离核的分裂以及珠心组织细胞的降解;在雌配子体细胞化过程中,雌配子体游离核区CaM含量的增加,推测可能与游离核细胞壁的形成相关。类Cyclin C蛋白伴随着油松雌配子体游离核的旺盛分裂而表达,在败育后期不育系胚珠中类Cyclin C蛋白过度表达,推测可能参与了油松游离核有丝分裂的调控,类Cyclin C蛋白异常表达可能是影响雌配子体育性的重要因素。更多还原

【Abstract】 The cones of clone 28 can aggrandize normally, but their ovules aborted due to the free nuclei mitosis failure of female gametophyte in the seed orchard of Pinus tabulaeformis Carr. in Xingcheng, Liaoning Province. It is an excellent material for mechanism research on the female gametophyte development of woody plants with its steady genetic characteristic of female sterility. The projective of this study was to analyze the mechanism of the free nuclei mitosis failure in this female sterility clone. After system studies of cytomorphology and histochemistry in ovules during the free nuclei mitosis failure, a fine cell culture from the embryos of P. tabulaeformis was established in this paper. The regulation mechanism of cell cycle in P. tabulaeformis was analyzed by cell synchronization, flow cytometric and 2-dimensional electrophores. Western blotting and immunohistochemical were applied to exploring the expression of the cell-cycle regulating factors during the free nuclei mitosis failure. The results were valuable information for understanding the expression and regulation of cell-cycle associated factors during female gametophyte development of gymnosperm, which were significant for extending study field of woody plants developmental biology and uncovering the mechanism of female sterility.1. The changes of the microstructure, ultrastructure and nutrient in the integument, female gametophyte and free nuclei in the clone 28 and clone 11 during the free nuclei mitosis failure were studied by isolation technique of female gametophyte, electron transmission microscope and histochemistry. The female gametophyte development in the female-sterile clone stopped at the stage of free nuclei. The cell organs of female gametophyte in the female-sterile clone were abnormal, the nucleus became swollen,the nucleolus disappeared, and the chromatin began to condense. There were large amounts of abnormal mitochondria swelling and extending in the cytoplasm of female gametophyte and their inside structures became unclean, while the number of vacuole decreased significantly even no larger vacuoles were found in the cytoplasm. Depression and rupture appeared in tonoplast. Cell walls were getting thick in nucellus cells near the female gametophyte and polysaccharides accumulated abnormally.2. The result of study on the cell cycle in P. tabulaeformis showed that suspension cells in exponential phase treated with 3 mM HU for 24h, after subcultured for 3h following the treatment, 90.71% cells were in phase G1; cultured for 27h after the removal, about 40% cells entered in phase G2, meanwhile the maximum number of cells in metaphase/anaphase was found. The changes of protein components between cells in 3h and 27h after the removal were analyzed by 2-dimensional electrophoresis. The result showed there were 11 proteins expressed only in phase G1 and 4 proteins expressed only in phase M. The volume of 7 proteins increased and the volume of 3 proteins decreased in phase M compared to proteins of cells in phase G1. These proteins distributed mainly in 18kDa/pI6.3-30kDa/pI7.5 and 60kDa/pI6.5-70kDa/pI7.2, and may be necessary for regulation of distinct phases in cell cycle.3. It was found that CaM and Cyclin C-like protein played an important role in the free nuclei mitosis of female gametophyte. The result showed that the expression of CaM and Cyclin C-like protein changed similarly throughout the cell cycle, the content of CaM and Cyclin C-like protein in phase G1 was little, and increased greatly in phase S, then in phase G2 the content decreased. After that, cells entered into next cell cycle, the content increased again in phase S. These results indicated that CaM and Cyclin C-like protein involved in the cell cycle regulation of P. tabulaeformis. The distribution of CaM,Cyclin C-like protein expressed the time-space-regulation specificity. At the stage of free nuclei vigorous division, the CaM distribution in the layers of big cells in integument decreased, however, the CaM distribution in the outboard cells in the integument increased obviously. Therefore, the integument was probably taken as a synthetic site or a accumulated and scattered site of CaM, and regulated free nuclei mitosis and cell degradation in nucellus. The content increase of CaM in free nuclei suggested to be related to cell wall formation during cell generating process in female gametophyte. Cyclin C-like protein expressed following free nuclei vigorous division. Overexpression of Cyclin C-like protein at the late stage of the abortion suggested it might involve in the regulation of free nuclei mitosis, and its aberrant expression may be an important factor influencing the female gametophyte fertility.更多还原