黄瓜RNA-seq分析流程与参数报告

This study investigates the molecular mechanisms by which exogenous nitrate nitrogen (NO₃⁻-N) modulates low-temperature stress tolerance in cucumber (Cucumis sativus L.) roots, combining transcriptomic and physiological analyses. Hydroponically grown ‘Xintaimici’ seedlings were exposed to three NO₃⁻-N concentrations (0 mM [Group A], 12 mM [Group B], and 24 mM [Group C]) under low-temperature stress (10/15°C). RNA-Seq and qRT-PCR analyses were performed on root samples collected at 4 and 12 days post-treatment, with physiological validation focused on Groups A and B due to minimal transcriptional divergence between Groups B and C (70 DEGs at day 12). In Group B, 12 mM NO₃⁻-N significantly upregulated key genes in phenylpropanoid biosynthesis (CsaV3_3G004000 [PAL] and CsaV3_1G032150 [C4H], 4.2- and 3.8-fold, respectively; p < 0.01), leading to flavonoid accumulation (2.17 mg·g⁻¹ by day 4, p < 0.05 vs. Group A), while downregulating linoleic acid metabolism genes (CsaV3_7G024950 [LOX2]), resulting in a 5.4% reduction in free fatty acids (p < 0.05). In contrast, nitrate deprivation (Group A) activated ribosomal pathways (2,317 DEGs, KEGG:ko03010 , p = 2.62×10⁻¹⁵) and oxidative stress responses (CsaV3_5G030310 [cytochrome P450], 3.5-fold upregulation), but failed to improve membrane stability. Co-expression analyses suggested that WRKY (CsaV3_2G035960) and bZIP transcription factors play a central role in regulating phenylpropanoid genes (ρ = 0.68), revealing a CBF-independent cold adaptation mechanism. These findings demonstrate that 12 mM NO₃⁻-N enhances cold tolerance through phenylpropanoid-mediated ROS scavenging and membrane stabilization, providing a molecular framework for precision nitrogen management in cucumber cultivation under low-temperature stress.