氮素作为植物生长发育重要营养元素，参与水稻生产的各个阶段。当前我国氮肥利用效率远远低于世界平均水平，提高我国农田氮肥利用效率迫在眉睫；而随着全球变暖的逐渐加剧，高温对水稻产量及氮素利用效率(Nitrogen Use Efficiency, NUE)产生显著负向影响，因此挖掘到作物耐高温氮高效基因意义重大。作为中国主要的水稻生产优势集中区，江苏省育成品种历史悠久，在前面的研究中发现从1950年到2010年水稻品种的氮素生理利用效率(Physiological Nitrogen Use Efficiency，PNUE)随育种年代一直上升，而近十年呈现下降的趋势。鉴于江苏省水稻种植区域纬度跨度较大因此推测1958-2016年间的水稻品种可能由于稻田氮肥施用量以及气候因子的逐年变化而产生基因型的差异，最终导致对氮素的响应差异。因此本文从江苏省1958-2016年的主栽水稻品种的基因差异表达上入手，尝试挖掘出能够提高水稻耐热性以及氮素利用效率的新基因，为未来育种提供候选基因。本文主要研究结果如下：

1.鉴定到可能参与水稻耐高温氮高效的新基因OsHTNE3（high temperature resistant and nitrogen efficient-3）。将15份江苏不同年代不同区域的主栽品种在高低氮处理下进行连续三年的田间试验，对其产量及氮素利用效率与温度进行关联分析，发现高温抑制了水稻产量的形成，造成水稻减产30%左右；并且降低水稻氮素利用效率；在不同氮处理下，高温胁迫下高氮处理的水稻产量损失较低，氮素利用效率降低的幅度也较低。对不同年代在氮素响应上的基因表达进行差异分析并且与氮素利用效率相关联后，鉴定到可能的耐高温氮高效基因OsHTNE3；对其进行生物信息学分析后发现其可能是一类线粒体精氨酸转运蛋白（mitochondrial arginine transporter BAC2），广泛分布于水稻的各个亚种，但是在籼稻和粳稻中基因分布差异达到了20%；其具有四个单倍型，在HAP1和HAP2中，其千粒重、粒长、粒宽等均有显著差异，说明存在潜在的育种价值；并且其在水稻整个生长发育阶段都有所表达，集中表达在生殖生长初期，尤其在花药开裂及子房形成阶段相对表达量最高。

2.OsHTNE3的表达受到硝态氮的诱导且可能参与根系发育。本论文鉴定到了一个可能参与水稻氮素利用效率及耐热性的基因OsHTNE3，设置不同氮素形态及不同浓度的处理，发现OsHTNE3在根系和地上部的表达均受到硝态氮诱导；将野生型与突变体材料进行水培，培养四周后对其根系生理性状进行观测，发现在发现htne3的根长在两种氮浓度下均显著短于野生型，平均缩短了30%以上；不定根的数量在低氮处理下没有显著差异，但在高氮下处理突变体材料的不定根数量显著少于野生型，减少了接近40%。除此之外，突变体材料的地上部长度和干重在低氮处理下与野生型无显著差异，但地上部长度在高氮处理下显著低于野生型，降低了10%左右。

3.OsHTNE3可能在高温下调控水稻产量形成和氮素利用。将突变体材料和野生型在高低氮处理下连续两年（2023-2024）进行田间农艺性状进行分析，发现2024年（高温）高低氮处理下突变体的水稻株高显著低于野生型，株高降低4%-7%；但是2023年（正常温度）突变体的株高与野生型无显著差异；进一步分析发现在2024年突变体在高低氮处理下Ⅰ、Ⅱ、Ⅲ节间距均低于野生型，降低幅度在4%-18%之间，最终导致株高降低。在产量方面，2023年突变体与野生型的产量均没有显著差异，但是在2024年高低氮处理下，突变体材料的产量均显著低于野生型,减产幅度达到50%-60%；统计发现在2024年突变体材料的分蘖数显著多于野生型，而在2023年则与野生型无显著性差异；2024年突变体的每穗粒数、结实率和千粒重在高低氮处理下均低于野生型，说明这是导致产量降低的因素；但是这些差异没有在正常温度年份（2023）体现，表明突变OsHTNE3后是在高温下导致水稻出现株高降低、产量下降的性状；对氮素利用进行分析后发现，发现在高温胁迫下，OsHTNE3突变体材料的氮积累量降低显著降低，降低了58%-72%，但浓度并没有明显降低，说明这种差异可能是由于在高温下产量的变化所导致的；在氮素利用效率相关指标在高温下也显著降低，正常温度上则没有发生明显变化，因此推测OsHTNE3对氮素的调控受到温度的影响。

4.推测OsHTNE3具有控籽粒形态发育的功能并影响高温下籽粒微量元素的积累。进一步对其籽粒形态进行观测，发现在不同氮处理下突变体的粒长显著长于野生型，增加了10%左右；粒宽显著低于野生型，长宽比也是显著大于野生型；在水稻扬花期时采取突变体和野生型的穗子进行观察，发现突变体的颖壳表面细胞较野生型有所伸长，细胞数量也有所降低。紧接着对其籽粒品质进行分析后发现突变体材料籽粒的蛋白含量和直链淀粉含量在高低氮处理下与野生型均没有显著差异，但是在消解值和胶稠度上均高于野生型，说明突变体材料的籽粒口感风味好于野生型；对其籽粒内的微量元素进行测定后发现，突变体籽粒的微量元素含量在正常温度下与野生型没有明显差异，但是在高温下显著降低，推测其功能缺失后，在高温胁迫下加剧了籽粒微量元素的损失。

Nitrogen, a crucial nutrient for plant growth and development, is involved in every stage of rice production. Currently, the nitrogen fertilizer utilization efficiency in China is far behind the world average, making it an urgent task to enhance nitrogen use efficiency in Chinese farmland. Additionally, with the intensification of global warming, high temperatures have a significant negative impact on rice yield and nitrogen use efficiency (NUE). Therefore, identifying genes that endow crops with high temperature resist and nitrogen efficient is of great significance.

Jiangsu, as a major region of rice producing in China, has an outstanding tradition of cultivating rice varieties. Previous studies have shown that in Jiangsu, with the evolution of rice varieties, the physiological nitrogen use efficiency (PNUE) of rice has been increasing from 1950-2010. It is hypothesized that the genotypic differences of rice varieties from 1958-2016 can be attributed to the main differences on PNUE and climatic factors in filed, which ultimately lead to differences in nitrogen response.Therefore, this study focuses on gene expression of major rice varieties cultivated in Jiangsu Province from 1958-2016, aiming to identify novel genes that can improve rice heat resistance and nitrogen use efficiency, possibility providing candidate genes for high temperature resist and nitrogen efficient rice breeding. The main research findings are as follows:

1. A novel gene, OsHTNE3 (high temperature resistant and nitrogen efficient - 3), potentially involved in high - temperature tolerance and high - nitrogen efficiency in rice, was identified. Fifteen major rice varieties from different regions and eras in Jiangsu were subjected to field experiment for three consecutive years under low and high nitrogen treatments. Correlation analysis between yield, nitrogen use efficiency, and temperature revealed that high temperatures inhibited rice grain yield formation, causing a reduction of approximately 30% in yield and decreasing nitrogen use efficiency. Under high - temperature stress, rice under high - nitrogen treatment suffered less yield loss and a smaller reduction in nitrogen use efficiency compared to that under low - nitrogen treatment. Through differential gene expression analysis related to nitrogen response in different eras and correlation with nitrogen use efficiency, OsHTNE3 was found out. Bioinformatics analysis indicated that it may be a mitochondrial arginine transporter (mitochondrial arginine transporter BAC2), widely distributed across various rice subspecies. However, there is a 20% difference in its distribution between indica and japonica rice. OsHTNE3 has four haplotypes, with significant differences in 1000 - grain weight, grain length, and grain width between HAP1 and HAP2, suggesting potential breeding value. It is expressed throughout the entire growth and development stages of rice, with exhibits peak expression during the early reproductive growth phase, with the highest relative expression levels observed particularly during the stages of anther dehiscence and ovary formation.

2. The expression of OsHTNE3 is induced by nitrate nitrogen and may be involved in root development. Having identified OsHTNE3 as a gene potentially related to nitrogen use efficiency and heat resistance in rice, this study investigated its response to nitrogen by conducting treatments with different nitrogen forms and concentrations. Results showed that the expression of OsHTNE3 in both roots and above - ground parts was induced by nitrate nitrogen. Hydroponic experiments were performed using WT and mutant materials. After four weeks of cultivation, root physiological trait observations revealed that the root length of the htne3 mutant was significantly shorter (by more than 30% on average) than that of the WT under both nitrogen concentrations. Under low - nitrogen treatment, there was no significant difference in the number of adventitious roots, while under high - nitrogen treatment, the mutant had nearly 40% fewer adventitious roots than the WT. Additionally, the above - ground length of the mutant was not significantly different from that of the WT under low - nitrogen treatment but was approximately 10% lower under high - nitrogen treatment. The dry weight of the mutant was comparable to that of the WT under low - nitrogen treatment but significantly lower under high - nitrogen treatment.

3. OsHTNE3 may regulate rice yield formation and nitrogen use under high - temperature conditions. To explore the impact of OsHTNE3 on rice growth and development, field agronomic trait analyses of mutant and WT materials were conducted under low and high nitrogen treatments for two consecutive years (2023 - 2024). In 2024 (a high - temperature year), the plant height of the mutant under both low and high nitrogen treatments was 4% - 7% lower than that of the WT, while no significant difference was observed in 2023 (a normal - temperature year). Further analysis showed that in 2024, the internode lengths of Ⅰ, Ⅱ, and Ⅲ nodes of the mutant were 4% - 18% shorter than those of the WT under both nitrogen treatments, contributing to the overall reduction in plant height. In terms of yield, there was no significant difference between the mutant and the WT in 2023. However, in 2024, the yield of the mutant was 50% - 60% lower than that of the WT under both low and high nitrogen treatments, likely due to decreases in the number of grains per panicle, seed setting rate, and 1000 - grain weight. These differences were not observed in 2023, indicating that the mutation of OsHTNE3 leads to decreased plant height and yield specifically under high - temperature conditions. Nitrogen utilization analysis showed that under high - temperature stress, the nitrogen accumulation in OsHTNE3 mutants decreased significantly (by 58% - 72%), while the nitrogen concentration remained relatively stable, suggesting that this difference is likely due to yield changes under high - temperature conditions. Nitrogen use efficiency - related indicators also significantly decreased under high - temperature conditions but remained unchanged at normal temperatures, indicating that the regulation of nitrogen by OsHTNE3 is temperature - dependent.

4. It is speculated that OsHTNE3 plays a role in regulating grain morphological development and affects the accumulation of trace elements in grains under high - temperature conditions. Based on the observed changes in yield and 1000 - grain weight of the mutant materials, this study further examined grain morphology. Under different nitrogen treatments, the grain length of the mutant was approximately 10% longer, the grain width was significantly narrower, and the length - width ratio was higher compared to the WT. Observations of spikelets during the heading stage showed that the glume surface cells of the mutant elongated and the cell number decreased compared to the WT. A preliminary analysis of grain quality revealed no significant differences in protein and amylose contents between the mutant and the WT under low and high nitrogen treatments. However, the mutant grains had higher disintegration values and gel consistencies, indicating better taste and flavor. Trace element measurements showed that there were no significant differences in trace element contents between the mutant and the WT at normal temperatures, but significant decreases were observed under high - temperature conditions. It is hypothesized that the loss of OsHTNE3 function exacerbates the loss of trace elements in grains under high - temperature stress.