The basic helix-loop-helix (bHLH) is the second-largest TF family in plants that play important roles in plant growth, development, and stress responses. In this study, a total of 100 bHLHs were identified using Hidden Markov Model profiles in the Nicotiana tabacum genome, clustered into 15 major groups (I–XV) based on their conserved domains and phylogenetic relationships. Group VIII genes were found to be the most abundant, with 27 NtbHLH members. The expansion of NtbHLHs in the genome was due to segmental and tandem duplication. The purifying selection was found to have an important role in the evolution of NtHLHs. Subsequent qRT-PCR validation of five selected genes from transcriptome data revealed that NtbHLH3.1, NtbHLH3.2, NtbHLH24, NtbHLH50, and NtbHLH59.2 have higher expressions at 12 and 24 h in comparison to 0 h (control) of chilling stress. The validated results demonstrated that NtbHLH3.2 and NtbHLH24 genes have 3 and fivefold higher expression at 12 h and 2 and threefold higher expression at 24 h than control plant, shows high sensitivity towards chilling stress. Moreover, the co-expression of positively correlated genes of NtbHLH3.2 and NtbHLH24 confirmed their functional significance in chilling stress response. Therefore, suggesting the importance of NtbHLH3.2 and NtbHLH24 genes in exerting control over the chilling stress responses in tobacco.

基本螺旋-环-螺旋 ( bHLH ) 是植物中第二大 TF 家族，在植物生长、发育和胁迫反应中发挥重要作用。在这项研究中，使用烟草基因组中的隐马尔可夫模型谱鉴定了总共 100 个 bHLH，根据它们的保守结构域和系统发育关系分为 15 个主要组（I-XV）。发现第 VIII 组基因是最丰富的，有 27个 NtbHLH成员。基因组中 NtbHLH 的扩增是由于节段和串联重复。发现纯化选择在 NtHLH 的进化中具有重要作用。随后对来自转录组数据的五个选定基因的 qRT-PCR 验证表明，与低温胁迫的 0 小时（对照）相比，NtbHLH3.1、NtbHLH3.2、NtbHLH24、NtbHLH50和NtbHLH59.2在 12 和 24 小时具有更高的表达。验证结果表明，与对照植物相比， NtbHLH3.2和NtbHLH24基因在 12 小时和 24 小时的表达量分别高出 3 和 5 倍和 2 倍和 3 倍，对低温胁迫具有高度敏感性。此外， NtbHLH3正相关基因的共表达。2 和NtbHLH24证实了它们在寒冷应激反应中的功能意义。因此，提示NtbHLH3.2和NtbHLH24的重要性控制烟草冷应激反应的基因。