【作者】 王文生；

【导师】 黎志康；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 盐害和干旱是影响水稻生长发育及产量的两个主要的非生物胁迫,因此通过水稻芯片研究在盐胁迫条件下全基因组的表达谱的变化,了解具有不同耐盐性的基因型在全基因组表达谱水平上的基因变化差异,对于发掘耐盐相关基因,了解基因之间的相互作用及遗传网络,了解耐盐相关的分子机理,并最终应用到实际水稻生产中提供了理论基础。DNA甲基化能够通过对基因组DNA上的胞嘧啶的共价修饰,从而对生物遗传信息进行表观遗传水平上的调控,其在植物体响应外界环境胁迫、调控基因的表达、稳定基因组等方面发挥重要作用。本研究利用本课题组培育的耐盐聚合系177-103以及其轮回亲本IR64为材料,从表型和全基因组的表达谱方面对耐盐聚合系的耐盐机理进行剖析。应用甲基化敏感性扩增多态性(MSAP)技术研究了具有不同抗旱性和耐盐性的水稻品种在不同发育时期和不同组织中干旱和盐胁迫对基因组DNA甲基化水平的影响,探讨了DNA甲基化在水稻响应干旱和盐胁迫过程中的作用。通过对聚合系群体进行耐盐筛选和重复的表型验证后,筛选出了一批耐盐性得到明显增强的耐盐聚合系,这些耐盐聚合系同轮回亲本IR64具有较高的遗传相似性,并且主要农艺性状和产量性状没有发生明显改变；通过对耐盐聚合系的耐盐相关表型及生理分析后得出,我们筛选出的耐盐聚合系,大部分是通过保持较低的叶片钠离子浓度和较低的钠离子转运等来降低盐害对水稻生长的抑制和毒害作用而提高其耐盐性。我们选择其中一个耐盐聚合系177-103以及其轮回亲本IR64进行了盐和盐+ABA处理,以了解盐胁迫以及ABA对具有不同耐盐性的基因型的表型和基因表达谱的影响。我们得出：盐胁迫时外施激素ABA能够通过提高叶片的相对含水量,降低地上部钠离子浓度以及保持较高的钾钠离子浓度比等来降低盐对水稻生长的抑制和毒害作用,能够同时提高轮回亲本IR64和耐盐聚合系177-103的耐盐性；通过全基因组表达谱分析可知：盐胁迫条件下,耐盐聚合系177-103中的差异表达基因多于轮回亲本IR64,177-103中特异上调表达的基因都是以胁迫相关的基因为主,IR64中的上调表达基因以转录相关基因为主；盐胁迫条件下差异表达基因具有很强的组织特异性,在叶片中差异表达的基因多于在根中差异表达基因,并且这些组织特异表达的基因的功能有较大差异；盐胁迫条件下,外施ABA能够引起很多基因表达的变化,其中叶片衰老相关蛋白,脱氧木酮糖磷酸盐激酶和DUF26结构域蛋白受ABA诱导并对于水稻耐盐性的提高有很大的贡献作用；在导入的DNA区段内发现了3个与植物耐盐性相关的重要基因,这些基因为透胁迫和钙离子依赖的ABA信号传导中起到重要作用的膜联蛋白Annexin,在拟南芥中被证明在葡萄糖信号传导和ABA生物合成中起重要作用的短链脱氢酶/还原酶SDR(Short-chain dehydrogenase/Reductase)和植物脂质转运蛋白等；通过比较发现,该实验所得到的差异表达基因能够与已经定位的效应较大的耐盐QTL有很好的对应,在前人定位的耐盐相关QTL区域发现了与耐盐相关的基因。干旱和盐害都能够诱导水稻全基因组范围的DNA甲基化状态的改变。由于干旱诱导的DNA去甲基化位点可以依据它们的可逆性分为两类,一类包括了大约70%的位点,这些位点只受到干旱胁迫诱导出现,胁迫去除后又恢复到胁迫前的甲基化状态,另一类包括了大约29%的位点,这些位点受到干旱胁迫诱导出现,即使在胁迫去除之后甲基化状态不发生改变；在水稻苗期根部组织中,盐胁迫会引起DNA甲基化水平的显著下降,而且在盐敏感品种IR29中DNA甲基化水平下降的程度比耐盐品种FL478更大;干旱和盐诱导的DNA甲基化/去甲基化具有很强的组织特异性和发育时期特异性。更多还原

【Abstract】 Abiotic stresses such as drought and salinity are very important factors limiting rice growth and productivity around the world. Affymetrix rice genome array containing 48,564 japonica and 1,260 indica sequences was used to analyze the gene expression pattern of rice responsive to salinity stress, try to elucidate the difference of genome-wide gene expression profiling of two contrasting rice genotypes in response to salt stress and to discover the salinity related genes and gene interaction and networks. Epigenetic mechanisms such as DNA methylation, plays a crucial role in regulating gene expression in plant responses to environmental stresses. The objectives of this study were:(1) to characterize the phenotypic and physiological traits associated with salinity tolerance (ST) pyramiding lines, (2) to understand the expression pattern of salinity tolerance pyramiding line and its recurrent parent IR64, (3) to understand the roles of DNA methylation in the epigenetic regulation of gene expression in rice responses and adaptation to drought and salinity stresses.A set of pyramiding lines with significantly improved salinity tolerance were selected by salinity screening of pyramiding population and phenotypic confirmation. The genetic background, morphological and yield traits of those ST pyramiding lines were highly similar with IR64. Most of the salinity tolerance pyramiding lines can improve their salinity tolerance through lower shoot Na+ concentration and lower root to shoot Na+ translocation et al, but there were two pyramiding lines 177-71 and 183-33 may have different mechanisms for salinity tolerance.A ST pyramiding line 177-103 and its recurrent parent IR64 were used for phenotypic and gene expression analysis under salinity and salinity +ABA conditions. We found that exogenous ABA under salinity condition improved rice salinity tolerance by increasing leaf relative water content, decreasing shoot Na+ concentration and root to shoot Na+ translocation. Under salinity condition, the number of differentially expressed genes (DEGs) in 177-103 was more than that in IR64, and most of up-regulated DEGs in 177-103 are response to stress. But in IR64, most of up-regulated DEGs are transcription related genes. The DEGs under salinity showed very strong tissue specificity, the number of DEGs in leaf was more than that in root. A lot of genes differentially expressed by exogenous ABA treatment under salinity condition, such as Leaf senescence protein,1-deoxy-D-xylulose 5-phosphate synthase 2 precursor and Protein of unknown function DUF26 were induced by ABA and contributed to salinity tolerance.We also found some important genes related with salinity in intrgression DNA regeions, such as Annexin, SDR (Short-chain dehydrogenase/Reductase) and plant liqid transferase proteion (LTP) et al.MSAP results indicate that drought and salinity could induce genomewide changes in DNA methylation/demethylation. Most of drought induced methylation/demethylation sites were of two major types distinguished by their reversibility, including 70% of methylation/demethylation sites at which drought induced epigenetic changes were reversed to their original status after recovery, and 29% of sites at which the drought induced DNA demethylation/methylation changes remain even after recovery. Significant decrease of DNA methylation induced by salt stress was specifically detected in roots at seedling stage, and the decreasing extent of DNA methylation in IR29 was more than that in FL478. The drought and salinity induced DNA methylation/demethylation alteration showed a significant level of developmental and tissue specificity.更多还原