氮素是植物必不可少的大量元素，它不仅作为营养物质参与植物的生长发育，而且还作为信号分子调控植株的形态建成。硝态氮作为大多数植物的主要氮源，其吸收、转运及代谢的调控机制已在多种植物中被仔细研究。在果树生长过程中，氮素供应水平的高低对植株的鲜重、干重、叶面积和生长速率等生理指标都有重要影响。同时，氮素的合理施用不仅可以达到“以氮增碳”的效果，还能增强果实的库活性。适量氮肥的供应不仅可以增加果实的产量，而且还可以提高果实的品质。但是，过多的氮素供应不仅会在土壤中大量积累造成富营养化，还会促进果树地上部生长过旺，使果树体内的C/N比例失调，抑制生殖生长，导致落花落果，降低了果实产量和品质。因此，研究果树氮素的吸收利用及其调控机制有重要的科学意义和实践价值。梨（Pyrus L.）是我国第三大果树，目前对其氮素吸收利用的生理特性和分子调控机制的了解还知之甚少。因此，本研究分析了不同浓度硝态氮对梨苗生长发育的影响，探讨了梨幼苗对硝态氮的吸收、转运及同化的调控网络，鉴定了梨慢速阴离子通道SLAC/SLAH（S-type Anion Channel）的功能特性以及调控机制，为提高梨树氮素利用效率提供理论依据，也为梨树氮高效利用的分子育种提供了基因资源，主要结果如下： 1、探讨了杜梨（Pyrus betulifolia Bunge）幼苗对不同氮水平响应的生理特性，结果显示:氮素缺乏或氮素过量不仅抑制梨苗的生长发育，而且还会影响植株根系的形态建成及叶片的表型症状。在缺氮条件下，梨苗出现叶片的黄化、根系硝酸还原酶活性的降低、叶绿素含量的降低，而且还刺激了根系的伸长生长。相反，在氮素过量供应条件下，叶片表现为暗绿色并出现了焦化现象、根系生长受到抑制，根系硝酸还原酶的活性降低，以及叶绿素含量的降低。此外，不同氮浓度的处理还影响根、茎、叶中氮素的含量，同时还影响了植株对其它矿质元素的吸收转运。总之，梨苗对氮素响应的生理特性分析为梨树诊断氮素供应水平提供了参照标准，也为后续研究提高梨树氮素利用率奠定了基础。 2、为研究梨树对硝酸根离子吸收转运的代谢通路，利用转录组测序技术分析梨苗根系对不同时间硝酸根离子处理的动态响应。研究结果显示‘砀山酥梨’（Pyrus bretschneideri Rehd.）幼苗根系经过硝酸盐饥饿及重新供应处理后，10273个差异表达基因被确定，同时它们被锚定在49个GO分组及18个KEGG通路上。此外，它们的共表达趋势可分为45种模式。在梨苗响应氮素饥饿及重新供应氮素的过程中，在KEGG通路上确定了15个与氮素代谢相关的差异表达基因，即1个NRT基因、2个NR基因、1个NiR基因、2个GDH 基因、6个GS基因和3个GOGAT基因。此外，还发现了449个与氮素代谢相关的转录因子，且它们属于35个不同的家族。最后，选取了14个与氮素代谢相关的差异表达基因，并通过实时荧光定量PCR对转录组测序的结果进行验证。这些结果为研究氮素代谢路径提供了基础数据。 3、慢速阴离子通道在植物生长发育过程中不仅参与阴离子转运（如硝酸根离子和氯离子等）、而且在非生物胁迫、激素信号转导等方面有着重要的功能。在蔷薇科物种中（苹果、梨、桃、草莓和梅）一共检索到21个SLAC/SLAH家族成员，共分为3个亚家族，分别为I、II和III。荧光定量分析结果显示，梨中的慢速阴离子通道家族基因在其各个组织部位都有表达。蛋白结构分析表明，梨中SLAC/SLAH家族成员含有10个跨膜结构域，亚细胞定位结果显示其定位于细胞膜。此外，功能回补试验表明PbrSLAH2/3-1能够成功缓解突变体slah3-3植株铵盐毒害的现象。总之，这些结果为进一步了解慢速阴离子通道家族的进化、表达和功能分析提供了重要的参考信息。 4、植物中钙依赖蛋白激酶（CPK）是钙离子传感器，在阴离子通道SLAC/SLAH磷酸化过程中起着重要的作用。本研究在梨中共筛选到43个CPK家族成员，被分为4个亚家族。复制模式分析表明全基因组复制和随机复制事件是梨CPK基因家族扩张的主要驱动力。酵母双杂交和双分子荧光互补实验证实PbrSLAH2/3-1与PbrCPK32可以互作。当PbrSLAH2/3-1与PbrCPK32或者AtCPK21共同在非洲爪蟾卵母细胞中表达时，卵母细胞在激光共聚焦显微镜的检测下细胞膜发出黄色荧光，并通过电压钳检测到了阴离子电流。离子选择性分析结果显示PbrSLAH2/3-1对硝酸根离子的渗透性远高于氯离子。总之，梨中慢速阴离子通道PbrSLAH2/3-1通过钙依赖蛋白激酶PbrCPK32的磷酸化作用参与硝酸根离子的转运。

Nitrogen, identified as an indispensable element in multiple biological processes of plants, not only participates in the growth and development stages as nutrients, but also acts as a signaling molecule to regulate the morphogenesis of plants. Nitrate nitrogen is the primary source for plants to absorb and utilize nitrogen, and the regulation mechanisms of absorption, transport and metabolism of it have been specifically studied in multiple plants. For fruit trees, the level of nitrogen supply significantly affects various physiology indexes including fresh weight, dry weight, leaf area and growth rate. Applying nitrogen to fruit trees based on scientific evidences not only enriches the content of carbon, but also extends the duration of growing development period by promoting the sink activity of fruits. It can also enhance the activity of the fruit bank and prolong the growth and development of the fruit. In addition, employing of scientific amount nitrogen fertilizers can offer crucial assistance for increasing the fruit yields and fortifying the fruit qualities. Nevertheless, excess of nitrogen supply brings negative effects on the growing and development as well as other significant functions of fruit plants. Applying nitrogen fertilizers in excessive amount leads to the massive accumulation of nitrogen, which causes eutrophication of soil. The vegetative growth of the fruit trees is significantly promoted by excessively applying nitrogen, which results in the disruption of the C/N ratio balance, and then inhibiting the reproductive growth. Consequently, it is of great scientific significance and practical value to penetrate the understandings for the absorption and utilization of nitrogen in fruit trees and related regulation mechanisms. The physiological characteristics and molecular regulation mechanism of nitrogen absorption and utilization of Pyrus L., which is the third largest yield fruit in China, are remaining highly unexplored. In this study, the effects of different concentrations of nitrate nitrogen on the growth and development of pear trees, as well as the regulation mechanism of pear seedlings absorbing, transporting and assimilating nitrates are analyzed; the functional characteristics and regulation mechanisms of S-type Anion Channel (SLAC/SLAH) are characterized. Taken them together, we offer a theoretical basis for improving the nitrogen use efficiency of pear trees, and provide genetic resources for molecular breeding of pear with efficient nitrogen utilization. The main results are as follows: 1. The growing development of the pear (Pyrus betulifolia Bunge) seedlings as well as their root-morphological traits and leaf phenotype are proved to be responsive to the changes of nitrogen in this study. To analyze the responding patterns of the plant to nitrogen of different concentrations, the pear seedlings are treated by nitrogen with five different concentrations. The results presented that the deficiency and the excess of the nitrogen have impacts on the morphogenesis of the roots and the phenotypic symptoms of the leaves, and they both can inhibit the growing development of the plant. Under the condition of nitrogen deficiency, the treated pear seedlings were observed with leaf etiolation and stimulation of root elongation, as well as decrease in nitrate reductase activity and chlorophyll content. Pear seedlings treated with excess nitrogen, nevertheless, had presented phenotypes of leaf coking and suppression of root elongation. Additionally, it is verified that the changes of nitrogen concentration impact the nitrogen contents as well as the absorption of mineral nutrients in plant tissues. Taken together, the physiological characteristics of pear seedlings responding to nitrogen provide a good reference standard for the diagnosis of nitrogen supply in pear, and lay a foundation for studying the improvement of nitrogen utilization rate of pear. 2. To gain a further understanding for the molecular mechanism in pear (Pyrus bretschneideri Rehd.) responsible for the regulation of nitrate transport and assimilation, RNA-seq was performed on samples with different nitrogen treatments. In analyzing the data acquired from the RNA-seq, after starvation treatment and re-supply of nitrate, the 10,273 genes of differential expressions were obtained and annotated to 49 GO terms, which can be classified into 45 clusters having co-expression trends and identified to be involved in 18 KEGG-defined significantly overrepresented pathways. Identified by the KEGG pathways, 15 genes of differential expressions including 1 NRT gene, 2 NR genes, 1 NiR gene, 2 GDH genes, 6 GS genes and 3 GOGAT genes are related to the nitrogen metabolism, and they significantly differentially expressed in response to nitrate starvation and a nitrate re-supply treatment. Furthermore, 449 transcription factors belonging to 35 different families were identified during the nitrate treatments. The expression patterns of 14 randomly selected genes of differentially expressed were verified by qRT-PCR. Taken together, these results and data provide valuable resources for investigating the genetics of the nitrogen metabolic pathways and improving nitrogen utilization efficiency in pear. 3. More than playing irreplaceable roles in anion transport pathways of plant (nitrate, chloride, etc.), S-type anion channels also function significantly in abiotic stress responses and hormone signaling of plants. In Rosaceae species, a total of 21 SLAC/SLAH genes were identified, which can be classified into three subfamilies based on the structural characteristics and phylogenetic analysis. Transcriptome data indicated that PbrSLAC/SLAH genes were located in all issues of the pear. As the transient expression and subcellular localization experiments demonstrate, PbrSLAC/SLAH genes are located on the plasma membrane in Arabidopsis protoplasts cells. Additionally, functional complementation experiments showed that PbrSLAH2/3-1 successfully alleviated the ammonium salt toxicity phenotype in Arabidopsis slah3-3 mutant. These results provide valuable information that help to gain further understandings of the evolution, expression and functions of the SLAC/SLAH gene family in higher plants. 4. Calcium-dependent protein kinase (CPK), characterized as a calcium ion sensor, plays crucial role in stimulating phosphorylation of SLAC/SLAH proteins by interaction. A total of 43 PbrCPKs were identified from pear genome and divided into 4 subfamilies according to the phylogenetic analysis. Duplication mode analysis showed that genome-wide replication (WGD) or dispersed replication was the main driving force in the expansion of this family. By carrying out the Yeast two-hybrid and bimolecular fluorescence complementation assays, it is confirmed that PbrSLAH2/3-1 interacts directly with PbrCPK32. When PbrSLAH2/3-1 was co-expressed with either the Arabidopsis calcium-dependent protein kinase (CPK) 21 or PbrCPK32 in Xenopus oocytes, yellow fluorescence was emitted from the oocytes, and the typical anion currents were recorded in the presence of extracellular NO3−. In addition, the electrophysiological results showed that PbrSLAH2/3-1 was more permeable to NO3− than Cl−. Accumulating these evidences, we suggest that PbrSLAH2/3-1 crossing-talk with PbrCPK32 probably participate in transporting of nitrate nutrition in pear.