【作者】 刘树楠；

【导师】 丁毅；

【作者基本信息】 武汉大学 ， 遗传学， 2009， 博士

【摘要】 植物雄性不育是水稻等农作物利用杂种优势的理论基础。在农作物遗传育种的研究领域中,一个基础性的研究课题就与水稻的雄性不育的有关。本文的研究是采用透射电子显微镜制片技术,以自主选育的云南紫稻和珍汕97为实验材料,比较观察了水稻花药发育的全过程以及两种水稻不育系花粉败育的不同特征；采用武汉大学自主开发研制的多光谱显微成像仪,首次将多光谱显微成像技术和细胞内生物大分子物质的定量分析研究结合起来,以珍汕97、紫稻、红莲、马协等四种不同的水稻胞质不育系及它们对应的保持系为材料,跟踪研究了水稻花药发育过程中,不育系和保持系花粉细胞内的多糖物质含量的动态变化规律。得到了如下实验结果：1、在小孢子发育的早期,云南紫稻不育系的小孢子细胞质中就出现了空腔,出现败育的迹象。和珍汕97不育系相比较,云南紫稻不育系败育的时期出现得更早,这说明云南紫稻不育系应该是一种新型的胞质不育类型,即为无花粉型败育。在云南紫稻不育系花药败育的时期,花粉囊壁的绒毡层细胞结构完整,细胞之间的界限分明,细胞胞质浓厚,没有出现败育的迹象。绒毡层细胞的正常发育,可能是引起云南紫稻不育系发生败育的原因之一。2、研究表明,在花药发育的过程中,保持系花粉细胞中多糖物质的多光谱曲线呈V字型,V字形的低谷出现在550nm-570nm波长范围内。不育系的花粉细胞在发生败育时,在420nm处多糖物质的多光谱曲线会出现显著的下降。这一显著的下降信号与水稻不育系花粉败育的时期相关联,可以作为判定水稻不育系败育时期的依据之一。3、在水稻保持系花药的发育过程中,花粉细胞中多糖物质含量的动态变化规律为：自花粉母细胞减数分裂到形成四分体细胞,花粉细胞中PAS反应染色较深,多糖颗粒小而丰富,花粉细胞中多糖物质的含量也稍有升高；在单核花粉细胞中,多糖颗粒大而显著,花粉细胞中多糖物质的含量开始降低；到了单核晚期,花粉细胞中多糖物质的含量达到最低值,在二核期细胞中,多糖颗粒开始融解,多糖含量升高；到成熟期,可育的水稻花粉发育成熟,在花粉粒中积累大量的淀粉颗粒,多糖物质(淀粉)含量也达到了最大值。4、与水稻保持系相比,不育系不仅在光谱曲线上,而且在多糖物质的含量上,均存在着很大的差别。在花粉母细胞和四分体时期,不育系细胞中多糖物质的含量稍高于保持系细胞中的含量,但它们之间差异不显著。在单核早期的花粉中,.红莲、马协、珍汕97不育系细胞中多糖物质的含量与保持系相比,它们之间的差异不显著,但是在紫稻不育系细胞中多糖含量明显高于保持系细胞中的含量。在单核边位期和二胞花粉期,红莲和马协不育系细胞中多糖物质的含量明显低于保持系细胞中的含量。到败育时期,败育花粉中多糖含量明显低于成熟花粉中的含量,且差异显著。此外,以黑三棱属的两个代表种(密序黑三棱(Sparganium glomeratum)和小黑三棱(Sparganium simplex)为研究材料,采用FIASCO磁珠富集法,开展了一些有关核基因组SSR引物开发的研究工作。合成了54对密序黑三棱基因组SSR引物,从中筛选出了13对有效的SSR引物,其中有9对引物为多态性SSR引物。利用这9对引物,我们在5份小黑三棱材料中进行了跨种检测,其中有7对SSR引物在种间扩增成功,另外2对引物没有得到扩增的产物。运用筛选到的9对多态性SSR引物,对采自东三省不同地区的45份密序黑三棱进行了种质资源的群体遗传学分析,结果表明,分布在相同区域的密序黑三棱材料聚类在一起,而分布在不同地域的材料则聚类在单独的分枝上。这些基础性的研究工作,对揭示黑三棱属植物的地理分布特点,物种的遗传多样性,以及遗传变异与进化的关系都具有一定的参考价值。更多还原

【Abstract】 Male sterility is the biological basis of heterosis utilization in crop breeding, especially in rice (Oryza sativa L.). Therefore, research on rice male sterility is a fundamental field of plant genetics and breeding. In this paper, we compared the developments of sterile anthers and the features of pollen abortion between Zhenshan 97 and YunNan Purple rice by electron microscope. In addition, for the first time, we investigated polysaccharides produced in anther development in both four cytoplasmic male sterility lines including Honglian, Maxie,Zhenshan 97 and zidao and their maintainer lines by the multi-spectralimaging and micro-quantitative analysis of big molecular substances. The results are mainly as follows.1. YunNan Purple rice was a new cytoplasmic male sterility line belonged to non-pollen type abortion. The vacuolated cytoplasm took place at the early stage of microspore development, showing a trend of abortion. Additionally, the abortion occurs earlier compared with that of CMS-Zhenshan 97 rice. At the stage of pollen abortion, tapetum cells are active, represented by thick cytoplasm and clear boundary. Our data suggested the abnormal development of tapetum cells was probably one of the reasons led to the cytoplasmic male sterility of YunNan Purple rice.2. In the maintainer lines, multi-spectral imaging of polysaccharides showed a V-shaped curve, with the lowest value appear in the wavelength range from 550 nm to 570 nm. When the anther of male sterility line became abortive, curves of polysaccharides showed a sudden decrease at wavelength of 420 nm. We found that this decrease was closely related to the time of pollen abortion of rice, which in turn can be applied to determine the abortion period of rice.3. Observations on anther polysaccharide dynamics change of rice maintainer lines revealed that pollen mother cells were stained strongly by Periodic Acid Schiff (PAS), with small but abundant grains during the process of pollen mother cell meiosis. At the same time, the amount of polysaccharides in the cells increased slightly. In contrast, polysaccharide grains of uninucleate-stage pollen were bigger, more distinctive and there was a decrease in the amount of polysaccharides. At the late stage of uninuleate cell, the amount of polysaccharides reached its lowest. At the binucleate-stage pollen, polysaccharide grains became diffused, resulting in an increase of polysaccharides. At maturity, fertile pollen developed and produced ample starch grains, together with a highest value of polysaccharides (starch) in pollen cells.4. Cytoplasmic male sterility line differed from its maintainer lines not only by multi-spectral curves but by the amount of polysaccharides. At both tetrad and pollen mother cell stages, the amount of polysaccharides in pollen cells of male sterility line was slightly higher than that of its maintainer lines. Experiments further revealed that there was no significantly difference between them. At the early microspore stage, the amount of polysaccharides of CMS Honglian, Maxie and Zhenshan 97 was similar to that of its maintainer lines, but the amount of polysaccharides of CMS Zidao was significantly higher than that of its maintainer line. At late microspore and bicellular pollen stage, the amount of polysaccharides of CMS Honglian and Maxie was significantly lower than that of its maintainer lines. At the abortive stage, the amount of polysaccharides in abortive pollens was significantly lower than that of mature pollens.In addition, we carried out an experiment on the development of SSR marker of nuclear genome of the two representative species of the genus Sparganium, namely S. glomeratum and S. simplex. By FIASCO, we developed 54 pairs of SSR marker of nuclear genome in the species Sparganium glomaratum. Among them,13 pairs of them are effective but only 9 pairs belonged to polymorphic Simple Sequence Repeat (SSR) markers. Furthermore, cross-species amplification in five individuals of S. simplex showed 7 of 9-pair markers was successful to the amplification. At last, nine pair SSR markers was applied to test for the genetic variation between 45 individuals of Sparganium glomaratum from different regions of the northeast China. The results revealed that the samples from the same gathering localities clustered into a group. These findings will contribute to the understanding of the genetic variation and evolution and distributional pattern of the genus Sparganium.更多还原