水稻PTR/NRT1基因家族在氮素营养运输中生物学功能的系统研究

Plant growth, productivity and seed yield depend on the efficient uptake, metabolism, and allocation of nutrients including nitrogen. Plants have evolved efficient and selective transport systems for nitrogen uptake and translocation within the plant to sustain development, growth, and finally reproduction. Development of genetic varieties with improved nitrogen use efficiency (NUE) is essential for sustainable agriculture. NUE can be divided into two parts, First, assimilation efficiency involves nitrogen (N) uptake and assimilation and second utilization efficiency involves N remobilization. Besides uptake of inorganic nitrogen molecules such as nitrate and ammonium by specific transporters, plants also take up organic nitrogen as a nitrogen source through their corresponding transporters in the form of amino acids, small peptides and proteins. Understanding the mechanisms regulating these processes is crucial for the improvement of NUE in crop plants. .Except N uptake by high affinity nitrate transporters (NRT2s), ammonium trnasporters (AMTs), and ammino acid transporters, PTR/NRT1 family contains low affinity nitrate transporters (NRT1) and di/tripeptide transporters (PTR) for nitrate or peptide uptake or translocation. NRT1s and PTRs belong to the same family. 84 NRT1/PTR homologs have been found in rice, but only 2 genes have been functional determinated so far. They was predicated to play roles in translocation of nitrogenous nutrients for N uptake from soil and remobilization within plant， and play roles in plant development and growth ,or in tolerance of stresses in plants..This proposal will study the functions of the 84 members of the rice PTR/NRT1 family in nitrogen uptake and remobilization in rice. The biological roles will be gotten from collection or construction of their mutants and RNAi rice or overexpression these genes. Their substrates of transportation will be gotten from expression in yeast mutants or in oocytes. Their expression profiles will be gotten by digital expression analysis, promoter-GUS analysis, GFP-fusion analysis, real-time RT-PCR and transcriptome. Their roles in nitrogen uptake, translocation and remobilization will be gotten by analysis of the changed expression of transgenic rices.Deep functional analysis will be taken for some members of rice PTR/NRT1 family, such as OsPTR2, OsPTR9, OsGT1 etc. Some marker-free transgenic rices of OsPTR9 will be constructed and analyzed for molecular breeding..From this study, preliminary biological functions, substrates of transportation, expression patterns, etc of rice PTR/NRT1 family will be achieved. Biological roles of some members will be gotten. Potential of some genes in molecular breeding will be evaluated. A marker-free overexpressing OsPTR9 rice will be evaluated for high NUE and it will be tested in controlled field. Roles of these genes in nitrogen translocation in rice will be systemic evaluated.

氮素营养的吸收和运输在植物生长发育、产量形成及抗逆中发挥重要作用，提高氮利用效率是农业可持续发展的保障之一。加深对水稻含氮营养吸收、同化、运输及再循环的理解，将为农业上提高水稻氮肥利用效率、培育氮高效利用的水稻新品种提供理论指导。PTR/NRT1家族指一类寡肽或低亲和力硝酸根运输基因，水稻基因组中有84个PTR/NRT1基因，但仅2个基因进行过功能研究。本项目将利用水稻突变库和生物信息数据库等，通过转基因改变表达水平，酵母功能互补或非洲爪蟾卵母细胞表达进行运输底物测试，各种营养或发育等条件下的转录分析，代谢物运输的非损伤微测等，系统研究水稻PTR/NRT1基因的生物学功能。明确PTR/NRT1基因在水稻含氮营养的吸收、运输和重新移动中的作用。获得参与水稻氮高效利用、品质形成的相关基因等。评价它们在水稻品种分子设计中的作用，或者提供分子育种一个选择指标，提供分子品种设计的中间育种材料等。

氮素营养的吸收和运输在植物生长发育、产量形成及抗逆中发挥重要作用，提高氮利用效率是农业可持续发展的保障之一。PTR/NRT1（NPF）家族指一类寡肽或低亲和力硝酸根运输基因。本项目系统鉴定了水稻基因组中的93个NPF基因，明确了3个基因（OsNPF7.2,OsNPF2.2和OsPTR9）的生物学功能，并进行分子设计育种的应用。. 主要的研究结果：（1）系统分析发现水稻基因组中93个NPF基因成员。（2）超表达OsPTR9可提高水稻的氮利用效率，水稻在低氮肥水平下可保持较高的产量。（3）异源爪蟾卵细胞表达表明OsNPF7.2与OsNPF2.2是低亲合硝酸根运输体。OsNPF7.2与OsNPF2.2分别定位于液泡膜与细胞膜，参与细胞内及细胞间长距离的硝酸根运输，并影响水稻的生长发育。（4）水稻新不育系“植A”与“植优523”获新品种权。杂交水稻新组合“植优701”于2016年通过广东省农作物品种委员会的审定。（5）发现水稻osa-miR171c在调节水稻营养生殖向生殖生长转变，oas-miR1848调节甾醇、油菜素内脂与蜡质合成中的作用。. 研究的主要科学与应用意义：（1）OsNPF7.2是第一个鉴定到的水稻参与液泡硝酸根运输的NPF基因，它对根中细胞内硝酸根的平衡有重要意义。而OsNPF2.2则参与了硝酸根由根向地上部分的运输，OsPTR9则可用于水稻氮高效的分子设计育种。（2）在进行基础研究的同时，进行分子设计育种，探讨相关鉴定到的基因用于分子设计育种的可能性。如OsPTR9的高表达可提高水稻的氮肥利用效率，可能有分子设计育种的应用前景。参与培育的水稻新品种（不育系与杂交组合）已应用于生产，其中植优523（母本为植A）已推广应用80多万亩。. 项目取得的主要成果有：发表标注SCI论文共8篇（其中第一标注3篇，总SCI影响因子约40），获授权专利1项和水稻新品种权2项。培育的水稻新品种“植优701”通过广东省品种审定且培育的水稻品种植优523已推广种植超过80万亩。培养博士后2名，博士2名，另1名博士生与硕士生进行后续研究。