【作者】 常玉晓；

【导师】 张启发； 吴昌银；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 创建插入突变体库并从突变体中分离插入元件的侧翼序列标签(Flanking Sequence Tags,FST),然后建立FST数据库是功能基因组学研究的重要策略之一本研究用TAIL-PCR的方法从水稻T-DNA插入突变体库中分离了7035条FST,丰富了水稻的突变体库FST资源,为反向遗传学在水稻功能基因组学研究中的应用奠定了基础。减数分裂是真核生物有性生殖过程中的重要环节,在生物的世代交替中扮演重要角色。虽然在过去的一、二十年间,人们通过对酵母和拟南芥等模式生物的研究,已经对这个过程的分子机制有了较为深入的了解,但是,水稻作为禾本科的重要模式植物和杂种优势成功利用的作物,我们对其育性调控和减数分裂的分子机制仍然知之甚少。本研究利用筛选水稻T-DNA插入突变体库获得的两个不育突变体克隆了两个参与水稻减数分裂的基因OsRPA1a和OsRPA2-3,得到的主要结果如下：1.在形态上,osrpa1a和osrpn2-3两个突变体除在育性方面有变异外,其它方面均无明显差别。通过RNAi抑制野生型中内源OsRPA1a或OSRPA2-3基因的表达可以使水稻的育性降低,而且将OsRPA1a或OsRPA2-3转入相应突变体后可以使育性恢复,这证明了我们克隆到的OsRPA1a和OsRPA2-3基因均是控制水稻育性的基因。2.Osrpa1a和osrpa2-3两个突变体的胚囊发育均在减数分裂时期开始出现异常导致核提前降解从而不能形成正常的功能大孢子,最终不能形成正常的“七胞八核”结构,取而代之的是一些降解的核的痕迹。3.在osrpa1a突变体花粉母细胞的减数分裂过程中,同源染色体可以配对、联会,并在终变期形成12个二价体,但是在中期Ⅰ之后有染色体断裂片段的出现,暗示配对的同源染色体中可能仍有未被修复的DNA损伤。4.Osrpa2-3突变体的同源染色体在减数分裂前期Ⅰ时可以配对和联会,但是交叉结(chiasmata)的形成受到严重影响导致终变期时形成部分二价体和一些单价体,但是,和osrpa1a突变体不同,osrpa2-3突变体的花粉母细胞中自始至终没有染色体断裂片段的出现,说明OsRPA2-3对减数分裂细胞中DNA的修复不是必需的。5.和野生型相比,osrpa1a突变体对MMC、MMS和UV等DNA诱变剂的敏感性均提高了,而osrpa2-3突变体对上述诱变剂的敏感性没有显著变化,说明OsRPA1a对体细胞中DNA的修复也是必需的；而OsRPA2-3对体细胞的DNA修复也不是必需的。6.在MMC和UV处理后,和野生型相比,osrpa1a突变体中DNA损伤出现的更早,积累的更多,这进一步证明了OsRPA1a对体细胞中DNA的修复是必需的。7. Osrpa1a和osrpa2-3两个突变体的有丝分裂均没有明显变化,说明OsRPA1a和OsRPA2-3可能没有参与DNA的复制或者不是DNA复制的必要成分。更多还原

【Abstract】 The creation of insertional mutant library and isolation of the flanking sequence tags (FST) of the insertion tags to construct an indexed FST database is one of the important strategies for gene cloning. In this research, we isolated 7035 FSTs from our T-DNA insetional mutant library by TAIL-PCR, which enciched the mutant FST source and will benefit the reverse genetics strategy in rice gene cloning.Meiosis is a central step during the sexual reproduction in eukaryotes and is essential for their metagenesis. Over the past decade with yeast and Arabidopsis leading the foray, we have witnessed an explosion in understanding of the mechanisms of the meiosis process; however, we still know little about the meiosis control mechanism in rice, quite a disproportion to its important status in both agricultural production and academic research. In this research, we conducted the functional analysis of OsRPA1a and OsRPA2-3, which participate in the meiosis process in rice, exploring two sterile mutants generated by T-DNA insertion; the main results are as follow:1. Both of osrpa1a and osrpa2-3 mutants are identical to wild type in plant morphology, but show sterile phenotype at mature stage. In addition, suppression of OsRPA1a or OsRPA2-3 expression in wild type phenocopies the osrpa1a or osrpa2-3 mutation, respectively. And transformation of callus homozygous for the mutants with the genomic fragment containing the entire OsRPA1a or OsRPA2-3 gene yielded the wild-type phenotype, respectively.. Taken together, these results confirmed that the sterility of mutants was caused by the loss of OsRPA1a or OsRPA2-3 function.2. Osrpa1a and osrpa2-3 mutants have the same defects in female gametophyte development at the megasporocyte meiosis stage, resulted the abnormal functional megaspore. At mature stage, the structure of the typical "seven cells and eight nuclei" in the wild type embryo sac were instead of the footprint of degenerated nucleate in osrpa1a and osrpa2-3 mutants.3. In osrpa1a mutants, homologous chromosomes pairing, synapsis and forming normal 12 bivalents in prophaseⅠas that of wild type, however, chromosome fragment presents in the pollen mother cells after metaphaseⅠ, which indicates there still exists unrepaired DNA damage in the PMCs in osrpa1a mutants.4. In osrpa2-3 mutants, homologous chromosomes achieve pairing and synapsis normally, but the formation of chiasmat decreased dramatically, leading to the presence of univalents at diakinesis. In contrast to osrpa1a mutants, osrpa2-3 mutants do not show chromosome fragment during the meiosis process.5. Compared with wild type plants, osrpa1a but not osrpa2-3 mutants show hypersensitivity to MMC, MMS and UV irradiation, which indicates the different requirement of OsRPA1a and OsRPA2-3 in DNA repair pathway.6. After treated with MMC and UV, there is more unrepaired DNA damage presents in osrpa1a mutants, moreover, the unrepaired DNA damage presents earlier than in wild type.7. Mitosis is unaffected in osrpa1a and osrpa2-3 mutants, which suggests that OsRPA1a and OsRPA2-3 may not participate in, or at least dispensable for, DNA replication in rice.更多还原