【作者】 张亮然；

【导师】 王台； 种康；

【作者基本信息】 中国科学院研究生院（植物研究所） ， 植物学， 2006， 博士

【摘要】 染色体黏着是有丝分裂和减数分裂的关键事件,是保证姊妹(或同源)染色体正确分离并分配到子细胞中的关键调控环节之一,它建立于细胞分裂前的S期将新复制的姊妹染色体紧密联系在一起。来自酵母的研究结果已经证明姊妹染色体之间的黏着是由多亚基的蛋白质复合体-黏着素所介导的。在芽殖酵母有丝分裂中,黏着素由Scc1,Scc3,Smc1和Smc3四个亚基组成。减数分裂黏着素的组成与有丝分裂中的相似,只是Scc1被其减数分裂特异的Rec8变体所替换。目前,已经从高等真核生物线虫,果蝇,人,鼠以及拟南芥中分离到了黏着素相关的基因,但是对于这些基因在高等真核生物特别是植物细胞分裂中的功能还知之甚少。即使在酵母中人们对于减数分裂和有丝分裂过程中有关染色体黏着与分离的许多基本问题仍然不清楚,而且许多现象表明减数分裂的详细机制在各种生物中存在重大差异。我们通过同源克隆的方法证明水稻(和拟南芥)基因组编码4个RAD21/REC8-like基因。这4个基因均以单拷贝存在,在核苷酸水平上没有相似性。它们所编码的蛋白质的相似性主要局限于其N-末端结构域和C-末端结构域。这4个蛋白质的中间区域没有(或者仅有极低的)相似性,但是中间区域都含有潜在的核定位信号,PEST序列,分离酶的识别序列以及多个磷酸化位点。半定量RT-PCR,原位杂交以及Western杂交结果显示这4个基因都在生殖器官中优势表达,但是它们在花发育过程中的表达动态是不同的。OsRAD21-1和OsRAD21-3都在减数分裂时期的颖花中表达量最高,但是OsRAD21-3还在成熟花粉中高表达;OsRAD21-4在减数分裂前的颖花中表达量最高;OsRAD21-2则在雌雄蕊形成时期表达最强,之后逐渐降低。这些结果暗示这4个基因的功能可能是不同的。免疫荧光定位分析表明,OsRad21-1和OsRad21-3特异地定位于有丝分裂的染色体上,其分布动态表明这两个蛋白可能都参与了有丝分裂姊妹染色体之间的黏着。由于水稻四个RAD21/REC8类基因中,只有OsRAD21-3在花粉发育过程中表达,同时水稻花粉的发育成熟要经过两次有丝分裂,推测OsRad21-3蛋白可能参与这两次有丝分裂过程姊妹染色体之间的黏着。OsRad21-4则特异地定位于减数分裂前间期到中期Ⅰ的染色体上,说明它可能特异地介导减数分裂过程姊妹染色体之间的黏着。与其它已知的Rad21/Rec8-like蛋白不同,不论在有丝分裂还是在减数分裂过程中,OsRad21-2蛋白都不定位于染色体上而是特异地定位在核仁中,并且它的动态变化与核仁重建和解体的动态规律在时间更多还原

【Abstract】 Sister chromatid cohesion is essential for accurate chromosome separation and segregation during meiosis and mitosis. Cohesion is established in the S phase, by a phylogenetically conserved multi-protein complex called cohesin, to link newly replicated sister chromatids together until removed from chromosomes in proper time. In budding yeast, cohesin at least includes Scc1, Scc3, Smc1 and Smc3 four subunits in mitosis, but Scc1 is replaced by its meiosis-specific variant, Rec8 in meiosis. At present, many genes associated with cohesin have been isolated from several higher eukaryotic organisms such as Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Mus musculus and Arabidopsis thaliana. However, functional analysis for these genes has not been widely carried out. And many basic issues about meiosis and mitosis are still unclear even in yeast. Furthermore there are great differences about the mechanisms of meiosis between different organisms.We have isolated all the four RAD21/REC8-like genes from Oryza sativa; and each of them exists in genome with a single copy. There is no similarity between them at nucleotide level. The similarity for the 4 polypeptides encoded by the 4 genes is mainly concentrated in their N- and C-terminal domains. Their middle sequences between the two conserved domains contain potential nuclear targeting motif, PEST motif, separase recognition site and many phosphorylation sites.Results from semi-quantitative RT-PCR, in situ hybridization and Western blotting have showed that the 4 genes have different expression dynamics during flower development although they all are expressed preferentially in reproductive organs. Both OsRAD21-1 mRNA and OsRAD21-3 mRNA have the highest accumulation amount in flowers at meiosis stage, but there is also high amount for OsRAD21-3 mRNA in pollen. The highest accumulation amount for OsRAD21-4 mRNA presents in flowers at pre-meiotic stage, however for OsRAD21-2 mRNA at更多还原