关键词: 烟草, NtWRKY65, 过表达, 低氮胁迫, 功能验证

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