【作者】 陈娟；

【导师】 张春霞；

【作者基本信息】 西北农林科技大学 ， 林学， 2023， 硕士

【摘要】 氮素是核酸、蛋白质、叶绿素和酶等物质的组成成分,是植物生长发育不可或缺的营养元素,保证着植物体内正常物质循环和能量代谢。无机氮是能够被植物直接吸收利用的主要氮素形态,铵态氮（NH₄⁺）则是其中主要形式之一。植物对NH₄⁺的吸收利用通常是由铵转运蛋白（Ammonium transporters,AMTs）介导。植物根系依赖铵转运蛋白汲取土壤中的铵态氮并将其转运至植株的各个器官和细胞。因此研究植物中铵转运蛋白的表达调控及功能等,对生产中提高氮素利用具有重要的意义。小叶杨（Populus simonii）具有耐盐碱、速生和适应性强等特点,是我国西北地区重要的杨树树种之一。本试验以小叶杨为研究材料,对小叶杨铵转运蛋白PsAMT1.1在转运NH₄⁺及其对氮素吸收利用方面的功能进行了验证,并且获得了过表达PsAMT1.1转基因杨树。对过表达PsAMT1.1株系进行了不同氮素水平处理（0.01 mM NH₄NO₃,1 mM NH₄NO₃,2 mM NH₄NO₃）,测定了氮素吸收代谢相关酶的活性及NH₄⁺和NO₃^-等相关生理指标,研究了小叶杨PsAMT1.1对氮素吸收和代谢的影响。主要研究结果如下:（1）小叶杨PsAMT1.1基因在幼叶、成熟叶、老叶、茎和根系等组织均有表达,在幼叶中的表达量最高。从小叶杨根系中克隆得到PsAMT1.1基因序列,CDS序列全长1503bp,编码501个氨基酸。小叶杨PsAMT1.1与毛果杨PtrAMT1.1的同源性高达98.47%。（2）在烟草表皮细胞瞬时表达PsAMT1.1基因,PsAMT1.1蛋白定位在细胞膜上,表明其在膜上具有活性,是膜转运蛋白。（3）在缺失铵转运蛋白的酵母突变体31019b中异源表达PsAMT1.1基因,结果表明其对NH₄⁺有转运能力。在低浓度NH₄⁺环境中PsAMT1.1恢复了酵母的生长活性。在拟南芥四缺体qko（缺失AtAMT1.1;AtAMT1.2;AtAMT1.3;AtAMT2.1）中异源表达PsAMT1.1基因,PsAMT1.1过表达的转基因拟南芥对NH₄⁺的吸收曲线符合饱和Michaelis-Menten方程拟合的双回归曲线,Km为79.27±9.75μM,表明PsAMT1.1是高亲和性的铵转运蛋白。（4）获得3个过表达PsAMT1.1转基因杨树株系。不同氮素水平培养过表达PsAMT1.1转基因杨树和野生型株系30天,发现低氮和中等氮条件下过表达PsAMT1.1转基因杨树较野生型净光合速率、叶绿素含量和茎增长量显著增加,促进杨树生长。高氮条件下转基因杨树茎、株高增长量和光合速率与野生型相比差异不显著。（5）与野生型相比,低氮和中等氮条件下过表达PsAMT1.1转基因杨树根系和叶片中的NO₃^-含量、NH₄⁺含量和总氮含量积累增加,根中NR、NiR、GS、GOGAT和GDH活性均增强促进了NH₄⁺的同化,有利于NH₄⁺代谢;低氮条件下根中可溶性糖含量增加而中等氮条件下无显著差异。高氮条件下,相较野生型过表达PsAMT1.1基因的杨树根系中NO₃^-和总氮含量积累降低,NR、GS、GOGAT和GDH活性增强,可溶性蛋白和可溶性糖含量增加;叶片中积累NO₃^-和NO₂^-降低,NR、GS活性也降低。综上所述,PsAMT1.1基因在低氮条件下对杨树的氮素吸收利用发挥极大的作用。更多还原

【Abstract】 Nitrogen is a component of nucleic acids,proteins,chlorophyll and enzymes,is an essential nutrient for plant growth and development,maintaining the normal material cycle and energy metabolism in plants.Plants mainly absorb inorganic nitrogen,and ammonium（NH₄⁺）is one of the main forms.The uptake of NH₄⁺by plants is mediated by ammonium transporters（AMTs）.The roots of plant rely on ammonium transporter proteins to take up ammonium from the soil and transport it to various organs and cells of the plant.Therefore,it is important to study the functions and regulation mechanisms of ammonium transporters to improve nitrogen utilization.Populus simonii is one of the major poplar species in northwest China because of its salinity tolerance,rapid growth and adaptability.In this study,we verified the function of ammonium transporter PsAMT1.1 in ammonium transport,nitrogen uptake and utilization in P.simonii and obtained PsAMT1.1 overexpression transgenic poplar.The PsAMT1.1 overexpression transgenic poplar lines were treated with different nitrogen levels（0.01 mM NH₄NO₃,1 mM NH₄NO₃,2 mM NH₄NO₃）.The activities of enzymes related to nitrogen uptake and metabolism and related physiological indicators such as NH₄⁺and NO₃^-were measured,and the effects of PsAMT1.1 on nitrogen uptake and metabolism were investigated in poplar.The main results were as follows:（1）The PsAMT1.1 gene was expressed in young leaves,mature leaves,old leaves,stems and root tissues of Populus simonii,with the highest expression in young leaves.The PsAMT1.1 gene was cloned from the root of P.simonii,and the length of coding sequence was1503bp,encoding 501 amino acids.The homology between PsAMT1.1 of P.simonii and PtrAMT1.1 of Populus trichocarpa was as high as 98.47%.（2）The PsAMT1.1 gene was transiently expressed in tobacco epidermal cells,and the PsAMT1.1 protein was localized on the plasma membrane,indicating that it is active at the membrane and is a membrane transporter protein.（3）Heterologous expression of the PsAMT1.1 gene in the yeast mutant 31019b,which is deficient in ammonium transporter,had the ability to transport NH₄⁺and restored yeast growth activity at low NH₄⁺concentrations;while heterologous expression of the PsAMT1.1 gene in the Arabidopsis quadruple deletion qko（deficient in AtAMT1.1;AtAMT1.2;AtAMT1.3;AtAMT2.1）,the NH₄⁺uptake kinetic curve conformed to a double regression curve fitted by the saturated Michaelis-Menten equation,with Km of 79.27±9.75μM,indicating that PsAMT1.1 is a high-affinity ammonium transporter.（4）Three PsAMT1.1 overexpression transgenic poplar lines were obtained and cultivated for 30 days at different nitrogen levels（0.01 mM NH₄NO₃,1 mM NH₄NO₃,2 mM NH₄NO₃）.The net photosynthetic rate,chlorophyll content and stem growth of the PsAMT1.1overexpression transgenic poplar significantly increased compared with the wild type under low and medium nitrogen conditions.The differences in stem and plant height growth and photosynthetic rate of transgenic poplars under high N conditions were not significant compared with the wild type.（5）Compared with the wild type,the accumulation of NO₃^-,NH₄⁺and total nitrogen contents in the roots and leaves of PsAMT1.1 overexpression poplar increased under low and medium nitrogen conditions,and NR,NiR,GS,GOGAT and GDH activities in the roots were enhanced to promote NH₄⁺assimilation and favor NH₄⁺metabolism;soluble sugar contents in the roots increased under low nitrogen conditions but not under medium nitrogen conditions.Under high nitrogen conditions,the roots of PsAMT1.1 overexpression poplar showed lower accumulation of NO₃^-and total nitrogen,increased NR,GS,GOGAT,and GDH activities,and increased soluble protein and soluble sugar content compared with wild type;the accumulation of NO₃^-and NO₂^-in leaves decreased,and the activities of NR and GS also reduced.In summary,PsAMT1.1 plays an important role in nitrogen uptake and utilization in poplar under low nitrogen conditions.更多还原