WGCNA Analysis and Identification of Key Genes in Tobacco in Response to Different Nitrogen Levels

Background :Nitrogen (N) supply directly impacts growth and quality in flue-cured tobacco. To decipher molecular responses to N gradients, we integrated transcriptomics and weighted gene co-expression network analysis (WGCNA) on leaves from four N treatments: 0 (inherent soil fertility), 60 (low), 105 (standard), and 150 kg/hm² (high). Results :Phenotypic analysis revealed dose-dependent increases in leaf nitrogen content with higher N application, accompanied by excessive vegetative growth and delayed maturity at 150 kg/hm². Transcriptome sequencing identified 47,216 genes, with differentially expressed genes (DEGs) increasing linearly with N levels (1,458–2,147 DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment highlighted nitrogen metabolism pathways, yielding 14 DEGs (11 in assimilation, 3 in transport). Weighted gene co-expression network analysis (WGCNA) uncovered two modules (lightcyan1 and black) strongly associated with N responses, harboring transcription factors NtERF11 (AP2/ERF), NtWRKY3 (WRKY), and NtSRM1 (MYB). Sub-network analysis within these modules identified five hub genes: NtGLN1-1, two uncharacterized genes, NtDFC , and NtGDSL. NtGDSL may enhance nitrogen use efficiency (NUE) through stress-responsive mechanisms, while NtDFC could integrate N signaling with developmental processes. These findings provide novel insights into N regulatory networks in flue-cured tobacco. Conclusions :This study reveals the effects of nitrogen application rates on flue-cured tobacco growth and gene expression. By identifying key transcription factors and genes regulating nitrogen metabolism, it provides a theoretical basis for dissecting nitrogen regulatory mechanisms, optimizing fertilization strategies, and improving nitrogen use efficiency in tobacco production. The experiment was conducted with the same phosphorus and potassium fertilization levels (phosphorus 105 kg/hm2, potassium 210 kg/hm2), employing four nitrogen fertilizer levels: no nitrogen fertilizer, 0 kg/hm2 (T1); nitrogen deficiency, 60 kg/hm2 (T2); standard application, 105 kg/hm2 (T3); and nitrogen excess, 150 kg/hm2 (T4).The experiment utilized a randomized complete block design with plot size of 66.7 m2, replicated three times.Standard float bed seedling raising method was employed, selecting vigorous tobacco seedlings, transplanted on April 28, 2022.Planting density was 16,492 plants/hm2 with row spacing of 1.10 m and plant spacing of 0.55 m, equivalent to 1100 plants/acre, with protective rows around.Only healthy and uniform four-leaf stage tobacco seedlings free from pests and diseases were selected for transplantation.For T3 treatment, fertilization ratio was N:P2O5= 1:1:2, with nitrogen provided as urea (Red Label, N ≥ 46.2%), phosphorus as calcium superphosphate (Red Star, P2O5 ≥ 16.0%), and potassium as potassium sulfate (Rubobuo, K2O ≥ 52.0%).Potassium fertilizer was applied at 210 kg/hm2, with nitrogen and potassium split between basal (70%) and topdressing (30%) applications. Basal fertilizer was applied before transplanting, and topdressing during the topping stage; all phosphorus fertilizer was used as basal at a rate of 105 kg/hm2.

背景：氮（Nitrogen）供给直接影响烤烟的生长与品质。为解析植物对氮梯度的分子响应，我们对4种氮素处理下的烤烟叶片整合了转录组学（transcriptomics）与加权基因共表达网络分析（weighted gene co-expression network analysis，WGCNA），4种处理分别为：0 kg/hm²（固有土壤肥力）、60 kg/hm²（低氮）、105 kg/hm²（标准氮）以及150 kg/hm²（高氮）。
结果：表型分析显示，叶片氮含量随施氮量增加呈剂量依赖性升高，且150 kg/hm²施氮水平下出现营养生长过旺、成熟期延迟的现象。转录组测序共鉴定到47216个基因，差异表达基因（differentially expressed genes，DEGs）的数量随氮素水平升高呈线性增长（1458~2147个）。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析显示，氮代谢通路显著富集，共鉴定到14个DEGs，其中11个参与氮同化过程，3个参与氮转运过程。加权基因共表达网络分析（WGCNA）筛选到两个与氮素响应显著相关的模块（浅青色1模块和黑色模块），其中包含转录因子NtERF11（AP2/ERF家族）、NtWRKY3（WRKY家族）以及NtSRM1（MYB家族）。对这两个模块进行子网分析，鉴定出5个核心基因：NtGLN1-1、2个功能未注释基因、NtDFC与NtGDSL。其中NtGDSL可能通过胁迫响应机制提升氮素利用效率（nitrogen use efficiency，NUE），而NtDFC可将氮信号通路与生长发育过程相整合。本研究结果为烤烟的氮素调控网络提供了全新的研究视角。
结论：本研究阐明了施氮量对烤烟生长及基因表达的调控效应。通过鉴定调控氮代谢的关键转录因子与功能基因，本研究为解析烤烟氮素调控机制、优化施肥策略以及提升烟草生产中的氮素利用效率提供了理论依据。本实验采用统一的磷、钾施肥水平（磷105 kg/hm²，钾210 kg/hm²），设置4个施氮梯度：无氮肥（0 kg/hm²，记为T1）、缺氮（60 kg/hm²，记为T2）、标准施氮（105 kg/hm²，记为T3）以及氮过量（150 kg/hm²，记为T4）。实验采用随机完全区组设计，小区面积为66.7 m²，设置3次生物学重复。采用标准漂浮育苗法培育烟苗，选取长势健壮的烟株于2022年4月28日进行移栽。种植密度为16492株/hm²，行距1.10 m，株距0.55 m，折合每亩1100株，四周设置保护行。仅选取无病虫害、长势均匀一致的四叶期健壮烟苗用于移栽。对于T3处理组，施肥比例为N:P₂O₅:K₂O=1:1:2，其中氮肥采用尿素（红标，氮含量≥46.2%），磷肥采用过磷酸钙（红星牌，五氧化二磷含量≥16.0%），钾肥采用硫酸钾（鲁北牌，氧化钾含量≥52.0%）。钾肥总施用量为210 kg/hm²，氮肥与钾肥均按70%基肥、30%追肥的比例施用：基肥于移栽前施加，追肥于打顶期施加；所有磷肥均作为基肥施用，施用量为105 kg/hm²。