【作者】 周时荣；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 突变体是研究基因表达与功能分析的良好试验材料,也是当前功能基因组学研究的研究热点之一。雄性不育突变体在植物的育种、遗传学、生殖生物学和分子生物学的研究中提供了很好的材料。为了更好地理解花粉发育的分子机制,有必要识别和分离涉及此过程各个阶段的基因,并对其进行功能分析。有关水稻半不育的报道多以籼粳杂种半不育为主,并提出了很多假说,但半不育的分子机制仍不清楚。究其原因,籼粳杂种半不育多是由籼粳间不亲和基因位点互作引起的,遗传基础较为复杂,不同的组合可能具有不同的不育位点,同时也往往受遗传背景的影响,研究起来较为困难,若能用稳定的半不育材料进行研究,阐明半不育产生的分子机制,对克服生产中遇到的半不育问题具有重要的借鉴意义。为此,本研究对稳定的半不育突变体材料w207-2进行了详细的细胞学分析并运用图位克隆的方法分离了花粉半不育基因,该结果为从分子水平上揭示水稻及其它禾本科作物半不育性的分子生物学基础具有重要的指导意义。本论文主要研究结论如下：1.通过对水稻半不育突变体w207-2的形态和细胞学观察,发现突变体的主要特征是：营养生长和花器官发育基本正常,自交结实率稳定在30%-40%之间,花粉育性稳定在50%左右,而雌配子育性正常。扫描和透射电镜观察表明,突变体中不育的花粉瘦瘪,且极度变形,花粉内含物空缺,无淀粉粒积累,花粉内壁缺失,外壁结构异常,覆盖层和基足层肥厚,基粒棒趋于解体。通过花粉粒计数,发现突变体每花药的花粉粒总数相当于野生型的78%,可育花粉总数相当于野生型的40%。另外,还观察到突变体花药开裂性差,造成开花时花粉散出量减少,使散落在柱头上的花粉粒总数减少,继而导致小穗育性降低,因此除了花粉半不育外,裂药性差也是造成结实率低的原因之一2.花药发育过程的半薄组织切片分析表明,突变体的花药壁发育正常,绒毡层和中层能够正常解体。细胞学观察表明：突变体在减数分裂中期Ⅰ有一定数量的单价体,在后期Ⅰ能观察到落后染色体和染色体桥,在后期Ⅱ也能观察到落后染色体,在四分体时期有微核形成。以上结果表明,突变的基因与减数分裂有关,因此本研究中的材料是一个研究水稻减数分裂分子机制的优良材料。3.选择花粉育性作为育性指标,用9600株w207-2/Dular F2分离群体中2100株纯合隐性半不育个体将该半不育基因pssl精细定位于CAPS标记L2和dCAPS标记L3之间,距两标记均为0.02cM。两标记位物理距离约为28kb,含有5个完整的开放阅读框。与野生型相比,突变体在KINESIN1-LIKE基因的编码区存在一个单碱基突变,引起了一个保守氨基酸的改变。确定该基因为pssl的候选基因,并进行了转基因功能互补验证。4.利用RACE结合RT-PCR技术获得了OsKINESIN-1基因的全长cDNA,利用RT-PCR分析结合OsKINESINl promoter::GUS的转基因水稻的染色观察结果显示,该基因在各个组织均有表达,但在穗中表达量较高,并且在幼穗的发育过程中有一个上调表达然后又降低表达的过程,其表达量的最高峰出现在减数分裂附近。利用洋葱表皮细胞进行的35S::OsKINESIN1-EYFP融合蛋白亚细胞定位结果显示,OsKINESIN1定位在细胞核中。利用大肠杆菌体外表达系统表达出了该蛋白,发现该蛋白具有微管结合的活性。更多还原

【Abstract】 Male-sterile mutations provide an ideal source material for a range of genetic and molecular biological studies of reproductive biology. In order to well apprehend the molecular mechanism of pollen development, all genes involved in this course developmental stage must be identified and characteried.Many studies on the hybrid semi-sterility between indica and japonica have been reported and many hypothesizes have been put forward, but the molecular mechanism of semi-sterility has been unknown. It is difficult to study this problem due to this hybrid semi-sterility causing by indica-japonica interaction. The hybrid genetic foundation is complex, so hybrid semi-sterility in different varietal combinations may be different sterility loci. If we can use a stable genetic semi-sterile cultivar to study the semi-sterile molecular mechanism, it would be a reference for overcoming the semi-sterility in practices.w207-2 is a stable mutant, and mutants are favorable materials for studying gene expression and function analysis, at the same time, analyzing mutants is also the development aspect for researching the functional genomes. The entire rice genome has been sequenced and publicly considered to be the genome of the model plant in grass, at the same time, there is synteny between rice and other grass genome. Therefore, there is an important use for reference to other grass plants through disclosing the biology basis in molecular levels.The main results were as follows:1. Through the morphological and cellular observation, we found the main character of the semi-sterility mutant is as follows:w207-2 is almost normal in vegetative and floral development. The most obvious difference between the mutant and wild-type is the fertility. Under normal conditions, the spikelet fertility of w207-2 is 30-40%, and the pollen fertility is about 50%, but the female gamete fertility was normal. We examined the wildtype and w207-2 pollens using scanning electron microscopy and transmission electron microscopy and found the sterile pollens in w207-2 have irregular shape and most of them are shrunken, which have no accumulations of starch granules, lack intine and have abnormal extine. Their tectum and foot layer is much plumper than the normal ones, and the columella tend to degenerate. We counted the total number of pollen grains per anther. On average, the pssl mutant has～78%total pollen grains equivalent to wild-type and the fertile pollen grains is equivalent to～40%of wild-type. Others, we found the anther of w207-2 was almost indehiscent, and the anther indehiscence of w207-2 was found to reduce the number of pollen grains dropped onto stigma per spikelet, thus reduced and induced the spikelet semi-sterility of w207-2. The pollen semi-sterility may be one of the causes that make the anther indehiscent.2. The anther transverse sections demonstrated that w207-2 anther wall development is normal, and the tapetum and the middle layers can degenerate normally. The DAPI staining revealed that most of the sterile pollen aborted in the uninucleate stage, but the abnormalities can be found ever since pollen meiosis stage:The w207-2 forms univalent at metaphaseⅠ, delayed chromosomes and chromosomal bridge can be found at anaphaseⅠ, delayed chromosome also can be found at anaphaseⅡ, and forms micronucleus at tetrad stage. These results indicate that parts of the chromosomes have abnormalities in chromosome pairing and chromosome movement, this is different from other meiosis mutants. So w207-2 is a good material for the study of the molecular mechanism of rice meiosis.3. According to pollen fertility,2100 recessive homozygous semi-sterile plants were selected from a large w207-2/Dular F2 population to identify the recombination between the pssl locus and the tightly linked molecular markers. Finally, pss1was mapped to the region between one CAPS L2 and one dCAPS L3 marker with the genetic distances of 0.02 cM, respectively. Both L2 and L3 markers were located on a same PAC clone P0470F10 with physical size of about 28 kb. Five open reading frames can be predicted by a sequence analysis of this fragment. We sequenced the entire region and found a single nucleotide convert in the coding region of the KINESIN1-LIKE gene, which caused change of a conserved amino acid in the KINESIN-1 protein family. So we presumed this gene should be candidate of PSS1, and carried on the complementary test by transgenic technology.4. We get the full-length cDNA of OsKINESIN-1 by the RACE and RT-PCR technology. We also analyzed the expression pattern by RT-PCR and GUS staining of the OsKINESIN1 promoter-GUS transgenic plants, we found that this gene is widely expressed in various organs, but the expression level is higher in panicle, and there is a obvious up regulate period during anther development, then the expression level goes down, the peak appears near the meiosis stage. Transient expression in onion epidermal cells showed the OsKINESINl-EYFP fusion protein is exclusively localized to nuclear. We expressed this protein by an E coli. expression system, and found this protein can bind to microtubule in vitro.更多还原