Abstract Sweet osmanthus (Osmanthus fragrans) is a widely used evergreen aromatic ornamental tree with poor tolerance to low temperatures. The basic helix-loop-helix (bHLH) transcription factor protein family, which is the second largest protein family in plants, has been found to play essential roles in the response to multiple abiotic and biotic stresses, especially cold stress. However, the bHLH gene family in sweet osmanthus has not yet been analyzed. In this study, 206 OfbHLH genes, which were classified into 25 subfamilies, were successfully identified from recently released whole-genome sequence data. Segmental duplication events had contributed mainly to the expansion of the OfbHLH gene family and all these duplicated gene pairs had experienced purifying selection. The transcriptome data exhibited different expression levels of OfbHLH genes in various tissues. Quantitative real-time PCR results indicated that the expression patterns of most IIIb, III(d + e), IVc, IX, and XII subfamilies could be up- or down-regulated under cold stress, and, among them, IIIb, III(d + e), and part of the XII subfamily participated in the response to multiple abiotic stresses. Six candidate OfbHLH genes (OfbHLH120/128/133/163/164/191), which were significantly up- or down-regulated during the cold stress process, were identified. Further studies indicated that OfbHLH120/128/163 had transcriptional activation activity and were located in the nucleus, implying that these genes are involved in the regulation of expression of cold tolerance genes. This study provides a solid foundation for the functional analysis of the OfbHLH gene family members involved in cold stress response and could be useful in the breeding of sweet osmanthus with improved cold tolerance.

中文翻译：

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桂花bHLH转录因子的全基因组鉴定及其响应非生物胁迫的表达分析

摘要 桂花（Osmanthus fragrans）是一种用途广泛的常绿芳香观赏树种，耐低温性差。基本螺旋-环-螺旋 (bHLH) 转录因子蛋白家族是植物中的第二大蛋白家族，已被发现在应对多种非生物和生物胁迫，尤其是冷胁迫中发挥重要作用。然而，尚未对甜桂花中的 bHLH 基因家族进行分析。在这项研究中，从最近发布的全基因组序列数据中成功鉴定了 206 个 OfbHLH 基因，这些基因被分为 25 个亚家族。节段重复事​​件主要促成了 OfbHLH 基因家族的扩展，所有这些重复的基因对都经历了纯化选择。转录组数据表现出不同组织中 OfbHLH 基因的不同表达水平。实时定量 PCR 结果表明，大多数 IIIb、III(d + e)、IVc、IX 和 XII 亚科的表达模式在冷胁迫下可上调或下调，其中 IIIb、III( d + e)，部分 XII 亚科参与了对多种非生物胁迫的响应。鉴定了在冷应激过程中显着上调或下调的六个候选OfbHLH基因（OfbHLH120/128/133/163/164/191）。进一步研究表明，OfbHLH120/128/163具有转录激活活性，位于细胞核内，表明这些基因参与了耐寒基因表达的调控。