【作者】 覃怀德；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 遗传学， 2010， 博士

【摘要】 种子休眠在植物界是一个非常普遍的现象,对植物躲避不良环境条件,维持生存繁衍起着重要作用。随着追求快速整齐出苗以获高产为目标的人工驯化过程导致了许多作物种子休眠减弱。种子休眠减弱甚丧失给农业生产带来了严重的问题,如水稻、小麦、玉米在种子发育后期遇到阴雨天气,很容易发生穗发芽,严重降低稻米品质和种子质量。对于水稻,由于杂交稻制、繁种过程中"920"(赤霉素)施用量的增加,穗发芽危害日益加重,一般估计,正常年份穗发芽率为5%左右,特殊年份可达20-30%,甚至更高。另一方面,休眠过强,又会导致种子不能整齐萌发,出苗参差不齐等问题。因此,深入研究水稻种子休眠分子机理,有助于培育适度休眠的品种,对水稻等作物生产具有十分重要的意义。本论文利用强休眠品种N22进行了如下研究：1.构建强休眠水稻品种N22发育25天到35天种子cDNA文库,得到的文库重组率为89%,平均插入长度1000bp左右,符合文库构建要求,测序分析了种子休眠后期基因表达的信息,为后续研究提供基础。2.水稻种子休眠与脱落酸(ABA)紧密相关,利用ABA信号传导中关键作用元件ABRE作诱饵,通过酵母单杂交筛选N22休眠cDNA文库,得到了与ABRE元件结合的阳性克隆。3.通过60Co辐照N22,从M1代开始,调查抽穗期,于抽穗后35天分单株收获,调查发芽率,筛选发芽率高的单株。至M5代,获得发芽率稳定在40%左右的突变体Q4646。应用基因芯片技术对Q4646与N22进行了基因表达比较分析,寻找两者差异表达的基因。结果检测到544个差异表达基因,涉及氨基酸代谢、碳代谢、逆境应答、激素相关等多种途径。其中,热击蛋白(HSPs)、bZIP类转录因子、细胞分裂素(Cytokinin).赤霉素(GA)、生长素(Auxin)、木葡聚糖内糖基转移酶/水解酶基因(XTH)、扩展蛋白基因(EXP)、α-淀粉酶基因综合调控种子休眠。进一步,结合种子休眠QTL研究结果,将差异表达的基因与休眠QTL区间进行对应分析,获得了与休眠区间相对应的差异表达基因,为后续克隆工作提供了基因表达信息。4.克隆拟南芥休眠相关基因AtHUB1和AtHUB2在水稻中的同源基因OsBRE1和OsBRE2。构建了RNA干扰(RNAi)载体,转化水稻进行初步功能分析。更多还原

【Abstract】 Seed dormancy is a state in which seeds do not germinate even under favorable environmental conditions. Seed dormancy is especially important for cereal crops because it is associated with pre-harvest sprouting (PHS) which frequently happens in China and Southeast Asia and causes great loss of yield and reduction of grain quality. Heavy PHS may occur to more than 5% of the rice in the field, and in some year may be up to 20-30% for hybrid rice. Seed dormancy is a complex trait controlled by polygenes and modified by the genetic background and environmental factors during late stage of ripening.There are many reports on the genetics of rice seed dormancy. To our knowledge, over fifty QTLs for seed dormancy were mapped to rice chromosomes.Despite of this, the molecular basis of seed dormancy was poorly understood. To identify the genes involved in seed dormancy, the researches are as followed:1. Seed dormancy cDNA library was constructed with the seed of N22 from 25 to 35 days after heading. The quality of the library was well with recombine rate of 89% and average length of 1000bp.The cDNA library contained the gene information of seed in the late stage of development.2. Abscisic Acid (ABA) play an important role in seed dormancy.With yeast-one-hybrid system, six clones were identified that can bind to ABRE element through selecting cDNA library.3. A weak seed dormancy mutant line Q4646 was derived by gamma-ray irradiation from the strong seed dormancy indica cultivar N22. The germination rate of Q4646 was more than 40%. Affymetrix GeneChip(?) Rice Genome Arrays were applied on these two lines using RNAs from seed of late stage of maturation and studied their global gene expression pattern differences. The results showed 544 genes were significantly different expressed. These genes were mainly involved in stress response, C-compound metabolism, plant development, DNA processing and lipid metabolism. Expecially, genes encoded heat shock proteins (HSPs), bZIP transcript factor, GA 20-oxidase 1, Cytokinin, Auxin, xyloglucan endotransglucosylase/hydrolase (XTH), Expansin(EXP), Alpha-amylase were significantly changed.These genes may play important roles in seed dormancy. Meanwhile, several differentially expressed genes can be mapped to previously reported seed dormancy QTL loci, suggesting that they may be candidates genes for seed dormancy. Our results provide useful clues for identified seed dormancy gene in future research.4.OsBRE1A and OsBRE1B were cloned that homologous to AtHUBl and AtHUB2. And RNA interference (RNAi) vectors were constructed and transgenic works were took to analysised their functions.更多还原