【作者】 周立国；

【导师】 罗利军；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 水稻是全世界最主要的粮食作物之一,但是水稻生产越来越受到水资源缺乏的限制。农业是用水大户,而水稻生产用水量占我国全部农业用水的70%,耗费了过多的淡水资源,这已经成为制约我国工农业发展的一个瓶颈。因此,开展对节水抗旱稻(WDR)研究是大势所趋。目前,虽然传统育种方法可以有效提高水稻抗旱性,但是对植物的抗旱机理了解不多,难以取得很大突破。由于植物的抗旱性十分复杂,涉及大量的基因表达与调控,必须不断发掘水稻抗旱相关基因,深入研究这些抗旱基因的功能,从而一方面明确水稻抗旱的分子机制,另一方面,为节水抗旱稻新品种选育提供基因资源。本研究首先对土壤中栽培的水稻进行自然断水处理,提取不同时期叶片中的总RNA；通过植物miRNA芯片和水稻全基因组mRNA表达芯片,扫描分析其与正常栽培水稻间的差异。对2个候选基因OsI17、OsDr1进行了克隆和表达分析。通过对超表达OsI17基因水稻植株的抗逆性鉴定,深入研究了OsI17基因的功能,明确了OsI17基因在水稻抗逆响应中的作用。本研究的主要结果如下：1.为了深入了解水稻抗旱机制,我们用植物miRNA芯片扫描水稻IRAT109在分蘖期和幼穗分化期干旱胁迫下miRNA变化情况。实验结果显示,在所鉴定的30个受干旱胁迫而发生变化的植物miRNA中,11个下调表达的miRNA如miR170,miR172, miR397, miR408, miR529, miR896, miR1030, miR1035, miR1050, miR1088和miR1126以及8个上调表达的miRNA如miR395,miR474,miR845,miR851,miR854,miR901,miR903和miR1125是首次发现受干旱胁迫诱导的植物miRNA; ath-miR170和ath-miR396家族都表现严格下调表达, ptc-miR474与ath-miR854a表现严格的上调表达。这些发现对于进一步揭示水稻抗旱的分子调控机制以及利用基因工程手段改善水稻的抗旱性具有重要的价值。2.运用水稻全基因组表达芯片分析了IRAT109在正常水分和干旱胁迫下基因表达的差异。我们发现了一系列与干旱胁迫响应有关的基础代谢途径,其中变化最明显的是碳固定途径。这显示了干旱胁迫下碳-水化合物合成代谢的适应性调节在水稻抵御干旱过程中起重要作用。3.采用qRT-PCR定量分析了候选基因OsDr1和OsI17基因在不同的非生物逆境处理下的表达谱变化,发现它们的表达受到ABA、水杨酸、茉莉酸、H202等激素,以及干旱、高盐、低温等逆境胁迫的诱导。4.应用RT-PCR方法克隆了OsI17基因含全长ORF的cDNA序列,并且构建了该基因的超表达、抑制表达等遗传转化载体,蛋白亚细胞定位载体,并导入到水稻中,获得了转基因材料。5.OsI17基因表达蛋白含有一个AT-hook和一个功能未知的保守结构域DUF296。DUF296是在原核生物和植物中广泛存在的保守结构域,其蛋白定位于细胞核,与核基质及染色质MARs区相结合。AT-hook模体和DUF296结构域都具备核定位功能。6.通过酵母双杂交实验验证,OsI17基因表达蛋白(或其DUF296结构域)具有同源蛋白结合功能,即OsI17蛋白可以通过DUF296结构域相互结合形成同源聚合物,发挥其结合及调整染色质构型的功能。7.以35S启动子诱导的OsI17基因超量表达转基因水稻,在干旱胁迫下其叶绿素含量提高,叶绿素a/b比值降低；转基因水稻叶片中POD比活力提高,植株对ROS的清除能力增强；旱胁迫下生长的转基因材料小维管束数目显著高于WT水稻(t检验,p<0.05),泡状细胞组数目也有所增加；离体叶片持水能力和叶片相对含水量提高。由此,OsI17基因提高了水稻的耐旱性和避旱性。8.全基因组表达芯片分析发现,在超表达OsI17转基因植株中,植株表达谱发生了变化。其中,变化最显著的代谢途径是PSⅡ系统,这一变化使植株在干旱胁迫条件下的光系统稳定性提高,为植株抵御逆境胁迫提供了更充足的物质和能量基础,从而改善了植株在水分胁迫下的生长状况。9通过对转基因植株苗期高盐、低温、干旱筛选,证明OsI17基因能明显增强水稻的抗逆性,提高水稻在高盐低温以及干旱等胁迫条件下的生存能力及胁迫后的恢复能力。成株期抗旱鉴定试验证明,与野生型水稻相比超量表达OsI17基因的水稻,结实率提高了17%～23%,单株产量提高了27%以上,生物学产量提高了25%～50%,百粒重增加了29%以上；根容量和干重明显提高,根系性状得到改善。10.OsI17基因启动子序列在野生稻和普通栽培稻中存在多样性。进化树分析表明了籼、粳稻以及野生稻之间可能的遗传进化关系,提示在野生稻材料中就已经开始出现籼、粳亚种的分化。11.用5’-RACE和3’-RACE方法,克隆了OsDr1基因的全长序列,确定了基因的转录起始和终止位点。定量实验表明,OsDr1基因受多种逆境诱导上升表达；受光照诱导和调节,并且其在24h光周期内呈节律性变化。更多还原

【Abstract】 Rice is one of the most important staple foods in the world. But rice production was confined by the lack of freshwater resources. As we know that, in our country agricultural production used too much freshwater resources, in which about 70% was consumed by rice. So studying the water-saving and drought resistance rice (WDR) was currently necessarily. At the present time, traditional breeding method is very significant effect in improving drought resistance of rice, but knows little about its molecular theory. As the complex truth of drought resistance theory, we must find and study more genes and gene network involved in drought response.In our study, we treated the rice that were planted in the soil with drought stress, then carried out a genome-wide profiling and analysis of miRNAs and mRNAs by microarray between drought-challenged rice and control. Two stress-responsive genes were studied for their expression levels under water stress. OsI17 gene was over-expressed in rice to test the ability on improving drought resistance. We found OsI17 gene could regulate the mRNA profiling and improve the drought resistance of rice, obviously. The main resalts are as follows:1. To gaina deep understanding of the mechanism of drought tolerance in rice, genome-wide profiling and analysis of miRNAs was carried out in drought-challenged rice across a wide range of developmental stages, from tillering to inflorescence formation, using a microarray platform. Among the miRNAs familes identified as significantly down- or up-regulated under the drought stress, miR170, miR172, miR397, miR408, miR529, miR896, miR1030, miR1035, miR1050, miR1088, and miR1126 were down-regulated miRNAs. miR395, miR474, miR845, miR851, miR854, miR901, miR903, and miR1125 were up-regulated miRNAs. miR156, miR168, miR170, miR171, miR172, miR319, miR396, miR397, and miR408 showed opposite expression to that observed in drought-stressed Arabidopsis. All of these revealed for the first time to be induced by drought stress in plants. The most conserved down-regulated miRNAs were ath-miR170, the miR171 family, and ath-miR396, and the most conserved up-regulated miRNAs were ptc-miR474 and ath-miR854a.2. We carried out a genome-wide profiling and analysis of mRNAs in drought-challenged rice by a microarray platform. A lot of metabolism pathways and genes were identified, which involved in drought-resistance of rice, in which the most important pathway was carbon fix. Maintaining a reasonable rate of synthesis of carbon-hydrogen compounds helps to enhance stress tolerance under drought-prone conditions3. Analyzed the expression profiling of OsDr1and OsI17 in rice. Up-regulated expression of OsDr1and OsI17 genes induced by various abiotic stresses (for e.g. Drought, salty, cold) and stress-related phytohormone (for e.g. ABA, JA, SA, and H2O2) treatments.4. We had cloned the full length cDNA of OsI17 in rice by RT-PCR method. Then we constructed the over-expression, antisense-RNA expression and OsI17-EGFP fuse vectors, and induced into Zhonghua11 (oryza sativa L.ssp. Japonica).6. OsI17 protein contains an AT-hook motif followed by a DUF296 domain, which was located in nucleus. AT-hook motif and DUF296 domain has nucleus location ability independently.7. The yeast two-hybrid testing showed that, the OsI17 protein (even if the DUF296 domain) had a homolog combining ability, that was necessary to playing its role.8. Under drought conditions, the transgenic rice has more content of total chlorophyll and lower chl a/b ratio than the WT. The drought stress testing at reproduce stage shows that, transgenic rice of OsI17 has higher POD activity, more small Vascular Bundles and bulliform cell groups in the flag leaf, higher RWC and Water-Retaining Capability of leaves. All of this shows that OsI17 enhanced the drought tolerance and drought avoidance of rice.9. Analysis of the transgenic rice profiling with microarray platform showed that, compared to WT,73 genes were up-regulated, and the PSII system was changed in evidence.10. The drought, salty and cold stress testing at seedling stage shows that, compared to the wild type, transgenic rice of OsI17 exhibited increased of stress tolerance. Compared to the wild type, the seed-setting rate, production per plant, biomass per plant and 100-seed weight of transgenic rice, exhibited 17-23%, more than 27%,25%～50% and more than 29% increase, respectively. The drought stress testing at reproduce stage shows that transgenic rice of OsI17 showed increased of drought tolerance.11. The OsI17 protein and the promoter all have sequences polymorphism among the cultivated rice varieties and wild rice lines (O.rufipogon). Phylogenetic tree of the OsI17 promoter showed that, the variety between japonica and indica had been exist among wild rice lines (O.rufipogon).12. The OsDr1 full length cDNA sequence was cloned by 5’-RACE and 3’-RACE method. OsDr1 expression was up-regulated by water stress, stress-related phytohormone; OsDr1 was expressed in circadian rhythms and regulated by light.更多还原