氮素协同调控水稻生育期和产量及氮素利用效率的机制

氮肥大量施用通常造成作物开花延迟和贪青晚熟，降低产量和氮肥利用效率（NUE），但其调控机制不清楚。在水稻中，我们已鉴定到了一个能同时直接调控抽穗开花时期和氮吸收利用的转录因子（NRF1）；获得了nrf1突变体恢复野生型表型和早熟、高NUE的遗传材料；筛选到了高氮导致开花和生育期延后、不变、提前的不同种质材料。在本项目中，我们将进一步通过回交、自交扩大其遗传群体，随后通过QTL/MutMap，GWAS等鉴定其突变和自然变异位点，获得候选基因。利用NRF1及其上下游基因和克隆的新基因的突变和超表达材料，分析它们协同调控生育期、产量、NUE中的分子机制，明晰水稻中同时存在的氮敏感和不敏感的开花调控途径。通过优良单倍型发掘、分子育种或基因编辑，获得生育期稳定、氮高效的水稻材料。研究结果将有助于突破“高产-稳产-氮高效”之间难以协调的技术瓶颈，服务于国家资源高效、绿色发展的重大需求。

Highly application of nitrogen (N) fertilizers commonly results in delay of flowering time, later maturation and decrease of yield and N use efficiency (NUE) in agriculture production, however, the molecular mechanism for the N-regulated flowering and developing is little known. .By screening the different responses of flowering time to N fertilization and transcriptome analysis of rice germplasms, we isolated a N-responsive transcription factor which belongs to MYB family with natural variations in rice germplasms. Knockout of this gene in the cultivar of Nipponbare delayed the flowering time (heading date) and maturation, particularly lost the response of flowering time to N fertilization. Therefore, we name it as N-Regulated-Flowering factor1 (NRF1). We have detected that inactivation of NRF1 in rice resulted in the transcriptional suppression of several N uptake and assimilation genes and inhibition of root acquisition of ammonium. By performing yeast two hybrid assay with cDNA library of the rice leaves harvested before the flowering transition, we have also obtained several candidate proteins which might be directly interacted with NRF1 at the flowering time in rice. In addition, we have screened the M2 generation of EMS mutated nrf1 lines and obtained the lines which restored to WT phenotype including the heading date/flowering time and developing stage. Moreover, we have isolated the representative materials from fully sequenced core rice germplasms which show the significant delay, no change, or early flowering (heading) and maturation, respectively, due to highly application of N fertilizers. By crossing with Nipponbare WT or the early maturation mutants or local high yield Wuyunjing cultivar, some BC1F1 and BC2F2 seeds of the selected materials have been generated for further analyses..Based on these promising rice materials and preliminary data, here, we propose to further characterize the molecular mechanism of the coordinated regulatory effects of NRF1 and its interacting proteins on flowering time, developing stage, N uptake and assimilation, and yield. The key genes which control the restoration of EMS treated nfr1 to WT phenotype, and the genes of natural variation in regulating flowering/developing stages by N fertilization will be cloned by QTL/MutMap and GWAS techniques. The single, double and multiple gene knockout mutants, as well as the complementation lines of NRF1 and its interacting or targeting genes will be generated for characterizing their physiological functions. The selected and hybridized lines of representative materials and the transgenic lines will be used to conduct the pot and field experiments with different N application levels in both subtropical area in summer (long day) and tropical area in winter (short day). The interacting effects of altered flowering/developing stage and N uptake/assimilation efficiency on rice yield and NUE will be characterized extensively. The molecular pathway of N-regulated flowering time (heading date) in rice will be drawn expectedly after this study. In addition, we will try to isolate the elite haploids of NRF1 and other new genes identified in this study. The molecular marker assisted selection or gene-editing techniques will be used to generate the ideal high NUE rice materials with relatively stable developing stages (entire growth duration) and high yield in planted area. This study will provide the novel theory and technique approaches for breeding the elite crops of high yield and NUE, which will be helpful for breaking the barriers among N fertilization - high NUE - high yield - stable yield in agriculture production.