烟草豫浓香201对低温胁迫的生理响应及转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0579

摘要/Abstract

摘要：

目的豫浓香201是利用0.6% EMS诱变中烟100自交6代选育获得的烤烟新品种，其抗病性好、品质优良，但大田表现低温敏感易早花特征，探究豫浓香201低温敏感形成的分子机制。方法以豫浓香201和中烟100为供试材料，4 ℃胁迫0、6和24 h观察其生长状态，测定H₂O₂含量、脯氨酸含量、抗氧化酶活性和内源ABA含量等生理指标，联合转录组学开展低温响应分子机制探究。结果与中烟100相比，4 ℃ 24 h豫浓香201严重萎蔫。低温处理后，中烟100内H₂O₂含量、脯氨酸含量、SOD和POD抗氧化酶明显升高，且持续低温内源ABA含量显著增加；豫浓香201细胞内H₂O₂含量极显著升高、脯氨酸含量、SOD和POD抗氧化酶无明显升高，且内源ABA含量明显降低。低温6 h，中烟100和豫浓香201参与低温响应的差异表达基因（DEGs）数目分别为4 314、3 769；低温24 h，中烟100和豫浓香201的DEGs分别为17 618、14 293。KEGG富集显示，低温胁迫下，两品种参与淀粉-蔗糖代谢通路的DEGs数量相当，中烟100 中DEGs参与植物激素转导通路较多，豫浓香201 DEGs参与植物昼夜节律通路较多。低温处理后，中烟100中可溶性糖相关基因SUS、TPP，α-淀粉酶基因AMY3，ABA通路相关基因SRK2E、SAPK3均上调表达，有助于增强抗寒性；豫浓香201中开花途径相关基因ELF3、FLK，冷调节基因COR27，光形态建成核心调控因子COP1与CBF负调节因子EIN3均表达上调。结论低温后，中烟100激活复杂而广泛的抗寒防御通路，而豫浓香201抗氧化酶系统调节能力不足，致使ROS清除系统减弱，对低温更敏感。

关键词: 烟草, 低温胁迫, 豫浓香201, 转录组学, 生理响应

Abstract:

Objective Yunongxiang 201 is a new flue-cured tobacco variety by self-crossing six generations, which was selected from the Zhongyan 100 0.6% EMS mutant library, with good resistance diseases and elite tobacco quality, whereas, it in field showed low-temperature sensitivity and early flowering characteristics. The molecular mechanism formed by low-temperature sensitivity of Yunongxiang 201 was studied. Method Yunongxiang 201 and Zhongyan 100 were used as test materials, their growth status was observed under 4 ℃ stress for 0 h, 6 h and 24 h. The physiological indexes of H₂O₂ content, proline content, antioxidant enzyme activity and endogenous ABA content was determined, and the growth status of plants recovered at 25 ℃ for 15 d was observed. The molecular mechanism was explored by combining transcriptomics. Result Compared with Zhongyan 100, Yunongxiang 201 wilted seriously at 4 ℃ for 24 h. Encountering low temperature, the H₂O₂ content, proline content, SOD and POD antioxidant enzymes in Zhongyan 100 increased significantly, and the content of endogenous ABA increased significantly under continuous low temperature; interestingly, the content of H₂O₂ in Yunongxiang 201 increased significantly, the content of proline, SOD and POD antioxidant enzymes didn’t increase significantly, and the content of endogenous ABA decreased significantly. The number of differential expressed genes (DEGs) involved in low- temperature-response of Zhongyan 100 and Yunongxiang 201 respectively were 4 314 and 3 769 at 6 h; Zhongyan 100 and Yunongxiang 201 respectively were 17 618 and 14 293 at 24 h. KEGG enrichment showed that the number of DEGs involved in starch-sucrose metabolism pathway was similar in the two varieties, Zhongyan 100's DEGs were more involved in plant hormone transduction pathway, and Yunongxiang 201’s DEGs were more involved in plant-circadian rhythm pathway under cold stress. After encountering low temperature, the expressions of soluble sugar related genes SUS, TPP, α-amylasegene AMY3, as well as ABA pathway-related genes SRK2E, SAPK3 were up-regulated in Zhongyan 100, which was conducive to enhancing cold resistance. The expressions of flowering pathway-related genes ELF3, FLK, cold-regulating gene COR27, constitutive photomorphogenic1 gene COP1 and CBF negative regulator EIN3 in Yunongxiang 201 was up-regulated. Conclusion After low temperature, the broad and complex cold-resistant defense pathway of Zhongyan 100 is activated, while the antioxidant enzyme system is insufficiently regulated, thus the ROS scavenging system were weakened, resulting in the more sensitive of Yunongxiang 201 to low temperature.

Key words: Nicotiana tabacum, cold stress, Yunongxiang 201, transcriptomics, physiological response

王亚乐, 李雪君, 孙计平, 孙焕. 烟草豫浓香201对低温胁迫的生理响应及转录组分析[J]. 生物技术通报, 2026, 42(2): 228-238.

WANG Ya-le, LI Xue-jun, SUN Ji-ping, SUN Huan. Physiological Response and Transcriptome Analysis of Tobacco Yunongxiang 201 to Low Temperature Stress[J]. Biotechnology Bulletin, 2026, 42(2): 228-238.

图/表 9

表1 RT-qPCR检测的引物序列

Table 1 Primer sequences detected by RT-qPCR

基因名称 Name	上游引物序列 Forward primer sequences（5′-3′）	下游引物序列 Reverse primer sequences（5′-3′）
NtFLK	TAGGGACGGTGCTCAATTCAA	GGACATTACCAGCGTCTTGC
NtEIN3	AGCCTCGTCAACAACCTAGC	TGTTGGTGTCCCCGAAGATG
NtSUS	AGTTCACCACTACAAGGGCAAGA	AGGGGAAAGGGTAGTGAGGTATT
NtTPP	GGATTCGGATGCTAAAGCGG	TACTTGTGATGAGTTGGAGATGA
NtTIFY10A	TGGAAACCAAACAACTACTACTATG	GTCATTTGTGCCTTTTCTGGTT
NtSAPK3	GAGGTCTTGGTAACTCCATCG	CACATATTTTTACACGTGGTGAAGC
NtCOP1	ACCTGGACAGGCAATAGCAAG	TCTTCAAGTCCCGCAGAATAAC
NtbHLH13	CGAGGATGGGGCTCAACAA	ATAACCACAAATGCTTACCCG
L25	CCCCTCACCACAGAGTCTGC	AAGGGTGTTGTTGTCCTCAATCTT

图1 豫浓香201和中烟100对低温的耐性差异A-C：豫浓香201和中烟100 4 ℃处理0（A）、6（B）和24 h （C）表型，标尺：2 cm；D：豫浓香201和中烟100 4 ℃处理前后生理指标图。不同大写字母表示极显著差异（P<0.01），不同小写字母表示显著差异（P<0.05）

Fig. 1 Different tolerance of Yunongxiang 201 and Zhongyan 100 to cold stressA-C: The phenotypes of 0 h (A), 6 h (B) and 24 h (C) at 4 ℃ in Yunongxiang 201 and Zhongyan 100. Bar=2 cm. D: Physiological index of Yunongxiang 201 and Zhongyan 100 before and after 4 ℃ treatment. Different uppercase letters indicate significant differences (P<0.01), different lowercase letters indicate significant differences (P＜0.05)

图2 差异表达基因的火山图和韦恩图

Fig. 2 Volcano and Venn maps of DEGs

图3 不同组间差异基因的KEGG富集分析

Fig. 3 KEGG enrichment analysis of DEGs among different groups

图4 豫浓香201和中烟100植物激素通路差异基因的表达分析A：植物激素通路不同时间点差异表达基因数目统计；B：不同时间点叶片ABA含量；C：KEGG植物激素通路注释图。红色表示基因上调表达，绿色表示基因下调表达，蓝色表示部分基因在0 h_vs_6 h存在上调或下调表达；信号通路每个节点左边代表豫浓香201（YN），右边代表中烟100（ZY）

Fig. 4 Expression analysis of DEGs in plant hormone pathway in Yunongxiang 201 and Zhongyan 100A: Statistics of DEGs in plant hormone pathway at different time points. B: ABA content in leaves at different time points. C: The map of KEGG in plant hormone pathway. Red indicates up-regulated expression gene, green indicates down-regulated expression gene, and blue indicates partial genes up or down-regulated expression at 0 h_vs_6 h. The left side indicates Yunongxiang 201(YN) and the right side indicates Zhongyan 100(ZY) at each node in plant hormone pathway

图5 豫浓香201和中烟100淀粉-蔗糖代谢通路相关酶基因的表达分析A：不同时间点差异表达基因数量统计；B：淀粉-蔗糖代谢通路相关酶基因的表达热图，热图中星号表示FDR<0.05，下同

Fig. 5 Expression analysis of enzyme genes related to starch-sucrose metabolic pathways in Yunongxiang 201 and Zhongyan 100A: Statistics of DEGs at different time points. B: Expression heat map of related enzyme genes in starch-sucrose metabolic pathway. Asterisk indicates FDR<0.05 in heat map, the same below

图6 豫浓香201和中烟100昼夜节律通路相关差异基因的富集

Fig. 6 Enrichment of DEGs in circadian rhythm-plant pathway in Yunongxiang 201 and Zhongyan 100

图7 差异表达基因的RT-qPCR验证

Fig. 7 Verification of DEGs by RT-qPCR

图8 参与耐冷性和开花进程的部分基因调控网络

Fig. 8 Part of the gene regulatory network involved in cold tolerance and flowering process

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