Abstract: In order to investigate the function of tobacco NtWRKY65 gene in response to low nitrogen stress，wild‑type K326（WT）and four transgenic lines with overexpressing NtWRKY65（OE）were used as materials，and the differences in phenotype，root traits，nitrogen metabolism related enzyme activity，nitrogen metabolism and aging related gene expression levels of different materials under low nitrogen stress and normal nitrogen supply conditions were studied by using hydroponic methods.The results showed that compared with WT plants，the main root length and lateral root number of OE plants significantly increased，and the dry matter mass and total nitrogen content significantly increased by 21.83%—45.12% and 94.65%—271.57%，respectively under low nitrogen stress.The growth potential of OE plants was significantly enhanced. Under low nitrogen stress，the activities of key enzymes for nitrogen absorption and assimilation（nitrate reductase，glutamate dehydrogenase），as well as the key enzyme for nitrogen redistribution（glutamine synthetase），and the expression levels of related genes（NtNR，NtGS1‑3，NtGS2，NtGDH1）in OE plants were significantly increased compared to WT plants.The expression levels of mature aging related genes NtMC，NtCP1，NtCP23，and NtCCD in the leaves of OE plants were significantly reduced compared to WT plants under low nitrogen stress.These results preliminarily confirm that overexpression of NtWRKY65 could promote root development in tobacco plants，enhance their ability to absorb，transport，and assimilate nitrogen under low nitrogen stress，inhibit leaf senescence，and positively regulate tobacco tolerance to low nitrogen stress. NtWRKY65 could be used as a candidate gene resource for genetic improvement of tobacco nitrogen use efficiency.

Key words: Tobacco, NtWRKY65, Overexpression, Low nitrogen stress, Functional validation

摘要： 为探究烟草NtWRKY65基因在低氮胁迫响应中的功能，以野生型K326（WT）与4 个过表达NtWRKY65 的转基因株系（OE）为材料，采用水培的方法研究了低氮胁迫和正常供氮条件下不同材料的表型、根系性状、氮代谢相关酶活性、氮代谢和衰老相关基因表达量的差异。结果表明，与WT植株相比，低氮胁迫下OE植株的主根长度和侧根数量显著增加，干质量和总氮含量分别显著提高21.83%~45.12%和94.65%~271.57%，OE植株的生长势明显增强；低氮胁迫下，OE植株的氮素吸收和同化的关键酶（硝酸还原酶、谷氨酰胺脱氢酶）、氮素同化和再分配关键酶（谷氨酰胺合成酶）的活性以及相关基因（NtNR、NtGS1-3、NtGS2、NtGDH1）的表达量均较WT植株显著提高；OE植株在低氮胁迫下叶片中成熟衰老相关基因NtMC、NtCP1、NtCP23和NtCCD的表达量均较WT植株显著降低。综上，过表达NtWRKY65能促进烟株根系发育，增强低氮胁迫下烟株对氮素的吸收、转运、同化能力，抑制叶片衰老，具有正向调控烟草耐低氮胁迫的功能，可以作为烟草氮素利用效率遗传改良的候选基因资源。

关键词: 烟草, NtWRKY65, 过表达, 低氮胁迫, 功能验证