小叶杨铵转运蛋白AMT1.2和AMT4.1基因的功能分析及其与抗盐性的关系

It is crucial for high nitrogen use efficiency to improve the capability of plant root to take up ammonium from soil (especially forest land). To further know the mechanism for high ammonium absorbing efficiency in Populus simonii and its relation to salt stress，functions of AMT1.2 and AMT4.1, both of which were highly expressed in root of Populus simonii and induced by salt stress, will be roundly studied in present project, and the following works will be done: (1) detect their expression characteristics via subcellular localization, qRT-PCR and in situ hybridization technique, (2) investigate their capability to transport ammonium as well as their characteristics in yeast system, (3) over-express or silence AMT1.2 and AMT4.1 in poplar to study the effect of expression level on nitrogen use efficiency and absorbing capbilities; (4) investigate the relationship between N metabolism and ammonium absorption and salt stress in root, and understand the roles of AMT1.2 and AMT4.1 in response to salt stress using transgenic plants. This study aims to understand the functions of AMT1.2 and AMT4.1 and relationship between high ammonium use efficiency and resistance to salt stress in Populus simonii. This study would give new insights into mechanism of ammonium uptake and its regulation. The results would serve to improvement and breeding for high nitrogen use efficiency in plant.

植物根系对铵态氮的吸收能力对于土壤（尤其林地）中氮的高效利用起着重要作用。在前期研究的基础上，本项目为进一步探明小叶杨铵态氮高效利用机制及其与抗盐性的关系，将研究细根中高度表达且受盐胁迫诱导的铵转运蛋白AMT1.2 和AMT4.1功能，通过亚细胞定位、定量PCR、免疫原位杂交研究二者在小叶杨不同组织细胞的表达特性；再分别在酵母突变体中异源表达，研究它们对铵态氮的转运能力及特性；进一步将AMT1.2 和AMT4.1在杨树中过量表达或干扰，研究其表达量改变对杨树N素吸收能力、生长及其抗盐性的影响；同时研究盐胁迫下小叶杨根系N素代谢与转运特点关系，以探明AMT1.2 和AMT4.1的功能及其与抗盐性的关系，为认识植物根系对土壤中铵态氮高效吸收转运机制，理解小叶杨抗贫瘠、抗盐与铵态氮的关系奠定基础。

植物根系对铵态氮的吸收能力对于土壤（尤其林地）中氮的高效利用起着重要作用。小叶杨是西北地区特有的耐瘠薄，耐盐碱，适应性强的杨树品种，为探明小叶杨铵态氮高效利用机制及其与抗盐性的关系，本项目鉴定了23个杨树铵转运蛋白基因家族成员并研究了其表达特性，研究了小叶杨在不同季节对氮素的吸收特点，以及盐胁迫和干旱胁迫下小叶杨对氮素吸收和同化特点以及铵转运蛋白对盐胁迫和干旱胁迫的响应，发现小叶杨AMT1.2和AMT4.1受盐胁迫的诱导表达上调，AMT1.2在小叶杨根系中特异表达，而AMT4.1在根系和叶片中均有较高的表达丰度。从小叶杨中克隆了AMT1.2和4.1基因，通过亚细胞定位研究表明AMT1.2和AMT4.1定位在细胞膜或者核膜上，为跨膜转运蛋白。在酵母系统中异源表达AMT1.2和AMT4.1基因表明其对铵态氮具有吸收转运能力，证实AMT1.2和AMT4.1均为铵转运蛋白。分离了AMT1.2启动子，构建过表达载体，蘸花法转化拟南芥，抗性筛选后获得纯合体，GUS染色结果表明AMT1.2在拟南芥根系中特异表达。AMT1.2 和AMT4.1在杨树中过量表达或干扰，经定量PCR检测和蛋白水平的检测获得了AMT1.2过表达株系，AMT4.1过表达和干扰株系，研究了转基因株系的氮素吸收特点和进行了抗盐性测定实验。AMT1.2过表达株系的生长优于野生型，氮素相关酶活性分析和含氮量测定等结果表明AMT1.2过表达杨树转基因株系吸收和同化氮的能力增强，证明AMT1.2在杨树氮素吸收中起着非常重要的作用。盐胁迫条件下AMT1.2过表达株系的生长也优于野生型，铵根离子的吸收增加，说明NH4+吸收可能与抗盐性有关。AMT4.1干扰株系的组培苗和野生型差异很小，移栽后发现苗子生长的时间越长株高差异越大，AMT4.1干扰株系的苗高显著低于野生型植株，光合、叶绿素含量等均低于野生型植株。野生型植株的氮素吸收能力高于AMT4.1干扰株系，从侧面证实了AMT4.1基因对氮素的吸收起着至关重要的作用。本研究探明了小叶杨氮素吸收和代谢特点，铵转运蛋白的表达特征，AMT1.2 和AMT4.1基因的功能及其与抗盐性的关系，为认识植物根系对土壤中铵态氮高效吸收转运机制，理解小叶杨抗贫瘠、抗盐与铵态氮的关系奠定基础。