【作者】 马雪峰；

【导师】 万建民；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 水稻是我国最重要的粮食作物之一，其产量的提高对确保我国农业可持续发展具有重要的战略意义。氮素是植物生长发育的主要营养元素，适当增加氮肥的施用量是水稻获得高产的重要措施，但是氮肥的过量施用也造成了作物种植效益降低和环境污染等负面影响。因此，研究如何提高作物的氮素利用效率对农业的可持续发展具有重要的意义。本研究利用一个水稻氮营养缺陷型突变体，通过图位克隆的方法分离了水稻中与氮代谢相关的一个重要基因，并进行了功能分析，为阐明水稻高效吸收利用氮素的遗传机理，从分子生物学途径改良作物的氮素利用率提供了重要的分子基础。本文主要研究结果如下：1．水稻突变体abp1来源于四倍体间杂种H3774的花药培养后代，是经过多代自交形成的稳定株系。主要特征是株高偏矮、穗长降低、穗上部坏死、干尖、籽粒窄细、千粒重低，最明显的差异是结实率低，只有10.26%，而野生型的结实率高达92.82%。虽然突变体的颖花发育正常，但花粉育性降低。氮肥梯度实验表明，突变体对外源氮素较敏感，随着氮肥施用量的增加，突变表型得到部分恢复。在相同的氮素供应条件下，突变体茎叶等营养器官中的氮含量与野生型相比增加显著，说明氮素向贮藏器官籽粒转运的效率比较低，是一个氮营养缺陷型突变体。2．之前的研究已经将水稻abp1突变基因定位在第4染色体的标记In10和AL1-1之间，物理距离为76Kb，含有13个完整的开放阅读框。测序比较发现，在ORF8的编码区有一个单碱基的突变，形成终止密码子（TGA），造成蛋白翻译的提前终止，因此将该基因确定为候选基因。将ORF8的全长cDNA和包含自身启动子的基因组片段分别转入突变体中，其转基因植株的表型得到恢复。另外，我们还分析了一个ORF8的T-DNA插入突变体abp1-2，它与abp1表型完全一致，表明ORF8就是目标基因，将其命名为OsARG。3．OsARG编码精氨酸酶，在水稻基因组中只有1个拷贝。定量RT-PCR和GUS组织染色的分析结果表明，该基因在水稻的根、茎、叶、叶鞘和穗中都有表达。突变体abp1中OsARG的表达水平与野生型相比显著降低，OsARG碱基突变还影响了氮代谢途径中的基因OsGS1;1的表达，与野生型相比，该基因的表达水平增加显著。亚细胞定位的结果初步表明，OsARG蛋白在水稻原生质体中呈点状分布，符合用软件分析得出的OsARG蛋白N端有一个线粒体定位序列的推测。4．在突变体abp1中，OsARG基因编码区的单碱基突变使得该蛋白翻译提前终止，造成了突变体中精氨酸酶的催化活性丧失，其作用底物精氨酸的含量过量累积，使abp1中氮素循环出现异常，从而导致了突变体株高偏矮、穗长缩短、穗上部坏死、籽粒窄细、结实率和千粒重降低等不良表型。5.在过表达OsARG的转基因植株中，单株产量、单株实粒数和结实率与对照相比都增加显著，而且产量的增加主要归因于单株实粒数的增加，表明利用OsARG基因的过量表达可以调控和改善植株体内的氮素代谢，提高水稻的氮素利用效率，实现增加产量的可能性。更多还原

【Abstract】 Rice is an important food crop and any genetic improvement on agronomic traits would playbeneficial roles on its yield increment. Nitrogen is considered as an essential major element forplant growth and development, and its reasonable application could bring the increase of grainyield. However, excessively applied nitrogen would decrease nitrogen use efficiency (NUE) andcause serious environmental pollution. From both view points of economic and environmentprotection, any measure on improving NUE at lower level of fertilizer supplement should beadopted. Through the study to an aberrant panicle mutant abp1, we detected out a mutationoccurred at the gene OsARG that responsible for nitrogen use efficiency, a single base pairmutation from G to T caused the abolishment of OsARG’s functions and excessive argnineaccumulation in such organs as stem, leaf and panicle, and finally resulted in the abnormalphenotype of abp1. Results from our experiment are summarized as follows:1. Compared with wild type plant, the mutant abp1exhibited shorter plant height and paniclelength, thinner grains and lightened1000-grain weight, as well as degenerated upper part ofpanicles, lower pollen fertility and seed-setting rate. By the exogenous nitrogen supplementexperiments, the abp1phenotypic defects could nearly be recovered to be normal along with theconcentration increment of applied nitrogen. At ripen stage, the nitrogen remained in stem andleaf were significantly higher than those in wild type. It suggested that certain amount of nitrogenhad not be transported out of stems and leaves. All results mentioned above suggested that abp1was a nitrogen nutrition related mutant.2. The abp1locus was previously mapped to the rice chromosome4between markers In10and AL1-1, spanning76kb physical distance in which13predicted open reading frames werefound. Sequence comparison between the mutant and wild type revealed a single base substitutionof G to T, introducing a new stop codon (TGA) at ORF8of abp1mutant. ORF8was subsequentlyconsidered as the likely candidate gene of abp1. The complement test transformed either cDNAor genomic fragment of ORF8into the abp1could recover abp1phenotype to be normal,especially, the panicle development fully restored. In addition, one T-DNA insertion to ORF8producing a similar phenotype as abp1provided an extra evidence that ORF8was responsible forthe mutant phenotype.3. ORF8was annotated as arginase encoding the gene OsARG which had only one copy inrice genome. Analysis of quantitative real-time PCR revealed that OsARG expressedconstitutively in various organs including root, stem, leaf blade, leaf sheath and panicle, whichwas further verified by GUS-staining analysis. In abp1, the OsARG expression level wasdecreased significantly. In addition, the expression level of OsGS1;1was increased, indicatingthat the mutation in OsARG had affected the transcript of other genes involved in the nitrogenmetabolism pathway. According to the result of GFP expression positions visualized by confocal microcope, OsARG exhibited spottedly distribution in protoplasts, which was corresponding tothe subcellular localization of mitochondria.4. In abp1, the mutation in OsARG caused the abolishment of OsARG’s functions andexcessive argnine accumulation in such organs as stem, leaf and panicle, which resulted in theabnormality of nitrogen metabolism, and finally resulted in the abnormal phenotype of abp1.5. In transgenic plants overexpressing OsARG, significant increases were observed in filledgrain number, seed-setting rate and the grain yield, and grain number was the yield componentthat was mostly responsible for the increase in grain yield with a strong relationship. Theseresults display a potential value for this gene utilization on improvement of nitrogen useefficiency.更多还原