草地早熟禾水氮互作下NRT家族基因调控机制的研究

Kenturky bluegrass is recognized worldwide as a good cool season turfgrass, and it's mainly used for city turf construction in northern China. Low nitrogen use efficiency and drought are the main factors which have limited the growth and ornamental quality of turgrasss. Thus, to solve the problem, exploring the nitrogen-fixation gene and drought-tolerant gene and breeding new varieties which can tolerant low nitrogen and drought conditions are important. Plant nitrate transporter protein has high, low, amphiphilic and NO3- absorb and transport system which can help the root system to absorb and transport NO3- and stimulate the plant to use nitrogen effectively. This paper studies the effects of water and nitrogen interaction on the regulation mechanism of NRT family genes through physiological and molecular biology techniques, mainly about the clone of NRT family genes in Kenturky bluegrass, the expression and regulation of NRT family genes in the water and nitrogen interaction and the preliminarily reveal of regulation mechanism of NRT family genes, the validation of mutual relationship of NRT family genes in the water and nitrogen interaction and the reveal of the relationship between NRTs proteins in the water and nitrogen interaction, function validation through over-expression in transgenic experiments, and the screen of NRT proteins which can not only tolerant drought conditions but also have high affinity through comprehensive analysis. The result of this study is of vital importance to the improvement of bluegrass's nitrogen use efficiency and water use efficiency, which provides the stress response mechanism of bluegrass to nitrogen and drought conditions.

草地早熟禾是世界公认优良冷季型草坪草，我国多用于北方城市草坪建植。氮素利用率低及干旱是制约草坪草生长及观赏质量的主要因素。挖掘固氮、抗旱基因资源，培育耐氮耐旱品种是解决问题的重要途径。植物硝酸盐转运蛋白（NRT）包含高、低及双亲和NO3-吸收转运系统，可有效调节根系吸收转运NO3-，使植物有效利用氮素。本研究采用生理和分子生物学技术，探索水氮互作中NRT家族基因调控机制。主要研究内容有草地早熟禾NRT家族基因克隆；NRT家族基因进行水氮互作下表达调控分析，初步揭示水氮通路中NRT的调节机制；NRT家族基因蛋白水氮互作下相互关系验证，揭示水氮互作中NRTs蛋白质间关系；通过转基因超量表达进行功能验证；综合分析筛选出水氮运互作既抗旱又高亲和硝酸盐转运蛋白基因。研究结果对提高草地早熟禾氮素利用效率及水分利用效率有重要意义，为完善草地早熟禾氮素营养及干旱胁迫应答机制，具有重要的研究价值。

草地早熟禾是优良的冷季型草坪，已成为北方园林绿化中重要的草种选择。氮素和水分是决定草坪草生长的重要因子，直接影响草坪质量，挖掘耐氮耐旱的基因资源尤为重要。本研究利用搜索NCBI中同源性较高物种的NRT基因序列和草地早熟禾转录组数据为依据，共获得2个NRT2高亲和硝酸盐转运蛋白和4个NRT1低亲和硝酸盐转运蛋白。NRT2.1和NRT2.4含有1个nitrate transmembrane transporter结构域，属于Nitrate/nitrite transporter NarK超级家族。NRT1.1，NRT1.2，protein NRT1/PTR FAMILY 5.8，protein NRT1/PTR FAMILY 4.3含有PTR2结构域，属于MFS Superfamily超级家族。利用实时荧光定量PCR分析发现NRT家族基因具有组织特异性，干旱，氮素及水氮互作处理下的表达水平中NRT2.1，NRT1.1，protein NRT1/PTR FAMILY 5.8等在逆境处理下表达量差异显著。低浓度的氮素浓度时,单一的硝态氮NO3-更有利于NRT2.1和NRT2.4基因的表达, 铵态氮NH4+可能抑制其表达。水氮互作相比单一低氮诱导促进NRT2.1基因的表达，却抑制NRT1.1和NRT2.4基因的表达。通过qRT-PCR及转录组测序分析，氮素浓度，干旱及水氮互作对于氮代谢相关酶基因的调控显著。GS1a，GS1b，NR，NRT1.1的表达水平随着氮浓度的增加，表达量随着增加。NRT2.1，GS2，Nir，NADH-GOGA,在低氮表达量最高，GDH在无氮素调控下表达量最高。NRT2.1的亚细胞定位分析，原生质体瞬时表达后NRT2.1-GFP定位于细胞核和细胞质中。建立了草地早熟禾再生体系，并构建NRT2.1，NRT1.1和protein NRT1/PTR FAMILY 5.8过表达载体，后续农杆菌转化草地早熟禾中。基于以上实验，本研究获得了耐氮耐旱的草地早熟禾NRT家族基因，为其应用提供理论基础，并丰富了草地早熟禾抗逆资源，为草地早熟禾耐氮耐旱品种的培养奠定基础。