【作者】 史金铭；

【导师】 孙野青；

【作者基本信息】 哈尔滨工业大学 ， 飞行器设计， 2010， 博士

【摘要】 为了探讨空间环境对植物种子产生生物学效应的机制,本论文从多个方面研究了空间飞行后当代水稻表型变化植株和非表型变化植株的表观遗传学效应,包括DNA甲基化多态性改变的程度及序列特征和遗传性,以及DNA甲基化变化与基因表达及基因组突变的关联。同时,通过对地基模拟辐射的研究剖析了空间环境中辐射因素对植物表观遗传学的影响。研究发现无论是空间飞行还是低剂量(2Gy)重离子辐射都能够引起水稻种子DNA甲基化的变化(P<0.05)。这种变化在表型变化植株上的程度明显高于非表型变化植株(P<0.01),而且DNA甲基化的变化更倾向于发生在基因组序列中的CNG位点上。DNA甲基化的多态性变化与基因组序列多态性变化的比较发现二者在变化率上具有相关性(P<0.001),即DNA甲基化变化明显的植株基因组序列变化也明显。序列特征研究表明空间飞行和重离子辐射引起的DNA甲基化变化广泛地分布在水稻基因组上包括基因外显子的区域上,但基因组序列多态性的变化则更倾向于发生在重复序列上。研究还发现部分发生在基因编码区的DNA甲基化和去甲基化变化与基因转录水平的下降或上升相对应。遗传性的分析表明无论是DNA甲基化的变化还是与甲基化变化相对应的基因转录水平的变化都能够遗传至下一代。以上结果说明表观遗传学机制参与了空间环境对植物种子的生物学效应,而且空间复合环境中的辐射因素可能是改变基因组甲基化的主要诱因。由于植物甲基化状态可影响基因表达并能稳定遗传,我们推测空间辐射引起植物种子当代出现表型变化且能稳定遗传的特征可能与基因组甲基化的变化相关。为了进一步研究辐射能够在水稻种子上产生明显表观遗传学效应的机制,本课题分析了种子与幼苗这两个不同发育阶段的水稻材料在相同剂量(2Gy)的重离子辐射后产生DNA甲基化改变的差异。结果发现种子的DNA甲基化变化程度明显高于幼苗(P=0.011)。为进一步分析这种特征与辐射诱因和抗逆生物学活性的关联,我们对比分析了0.02~20Gy剂量的重离子辐射引起水稻干种子、湿种子和幼苗的细胞生物学和抗氧化应激系统改变的特征。研究结果显示,当剂量小于2Gy时辐射能够对植物的生长产生刺激作用,而当剂量大于2Gy时则开始出现明显的抑制作用。三种不同时期水稻材料的比较发现低剂量辐射的生物学刺激效应和植物材料的含水量有关。染色体畸变的观察显示在三种水稻材料中染色体的畸变率都随着辐射剂量的增加而增加。但是,在较低剂量下(小于2Gy)水稻种子的染色体损伤要多于幼苗。丙二醛(MDA)的含量检测和两种抗氧化酶:超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活力检测结果表明,较低剂量(小于2Gy)辐射后,种子中的抗氧化酶的活力低于幼苗,MDA的含量高于幼苗;当辐射剂量大于2Gy时,幼苗中出现显著的抗氧化酶SOD和CAT活力下降和相应的MDA含量升高,与此同时染色体损伤也明显增加,存活率下降。这一结果说明低剂量的辐射生物学效应不但与辐射因素相关,还与生物体的抗氧化应激状态有非常重要的关联。抗氧化系统不能有效地激活很可能是种子对低剂量重离子辐射的敏感性高于幼苗的主要原因。更多还原

【Abstract】 In order to study the mechanism of biological effects of space environment on plant seeds, in the present dissertation, we investigated the space flight induced epigenetic effects on rice plant with or without phenotypic mutations on many aspects including: level of polymorphic DNA methylation changes, character of genome sequences with DNA methylation changes, inheritance of polymorphic site and the relationship between DNA methylation changes with gene expression and genomic mutations. At the same time, we detected the epigenetic effects of on-ground radiation experiment to analysis the role of radiation in space environment induced biological effects. Results show that DNA methylation changes can be induced on rice by both space flight and low-dose (2Gy) heavy ion irradiation (P<0.05). The frequencies of DNA methylation changes on phenotypic mutants are obviously higher than that on normal plants (P<0.01). Moreover, DNA methylation changes are prone to happen on CNG sequences on rice genome. We also found the correlations of the polymorphic rate between DNA methylation and genomic sequence alterations (P<0.001). Higher level of DNA methylation changes and genomic sequence changes often appear on the same individuals. Sequencing of the polymorphic sequences show that space flight and heavy ion radiation induced DNA methylation changes are widely spread on rice genomes including coding regions, while changes of genomic sequence are more prone to appear on repeat regions. We also prove that the hyper-methylation or hypo-metylation changes on coding genes are corresponding to the increase and decrease of the expression of the gene. Both the altered DNA methylation patterns and the expression level changes are heritable. These results strongly suggest that epigenetic mechanisms are involved in the biological effects on rice seeds induced by space environment. Moreover, the radiation factor may be the main mutagen in the complex pressure in space. The heritable DNA methylation changes are related to gene expression, so we speculate that DNA methylation changes may be a reason of space radiation induced heritable phenotypic mutants in the first generation after treatment.In this study, we also compared the DNA methylation changes on different life stages of rice (seed and seedling) under the heavy ion radiation at same dose (2Gy) to explain the mechanism of the high level epigenetic changes on seeds of plant. The results show that frequencies of DNA methylation changes on rice seeds are higher than that on seedlings (P=0.011). Observations on chromosomal aberrations show that the frequencies of chromosomal aberrations were increased with the increasing dose in all of the three rice materials. More chromosomal damages were observed in seeds than seedlings at dose lower than 2Gy. Detect of malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD) and catalase (CAT) also show that the activities of SOD and CAT in seeds are lower than that in seedlings at dose lower than 2Gy. The content of MDA in seeds is higher than in seedlings at dose lower than 2Gy. At dose higher than 2Gy, obvious increase of content of MDA and decrease of activity of SOD and CAT together with the high level of chromosomal damages and lethal effects are observed in seedlings. Our results prove that the biological effects of radiation are not only related to the kind of radiation, but also related to the ability of antioxidant systems of biological materials. We suggest that the higher radiosensitivity to IR induced chromosomal damages of non-growing seeds than growing seedlings at lower dose may be because of the inactive antioxidant systems in seeds.更多还原