The basic helix loop helix (bHLH) transcription factor comprises one of the largest plant-specific transcriptional regulators in plant growth and development that response to biotic and abiotic stresses. Many members of bHLH play essential roles in the growth of root hair and response to drought, salt, and cold stresses. The family of bHLH genes has been found in many species; nevertheless, the barrel medic and alfalfa species still have a minute gap of bHLH new members thus far. This research aims to identify members of the bHLH family in barrel medic and alfalfa and elucidate their expression pattern level, network analysis, predictive 3D modeling and phylogenetic relationships. Here, we identified and characterized the bHLH gene family in both barrel medic and alfalfa plants and their genes expression response to drought, salinity, and cold stresses. A total of 159 MtbHLH and 133 MsbHLH genes were identified and characterized, divided into 18 subgroups and 17 subgroups, respectively. As a ubiquitous and popular method, neighbor-joining clustering was used. Based on the phylogenetic analyses, the VIII and IX subfamily and X subfamily were selected as the stress-related subfamily in these two species. The 154 MtbHLH genes were progressively distributed on the 8 chromosomes and 23 tandem duplicated genes, and 44 duplicated genes segments were detected in MtbHLH family. The analyses of gene ontology discovered the bHLH predominantly functions in protein and DNA binding in these two species. The results of Ka/Ks were < 1, which showed that the most orthologous of the bHLH gene values was found between A. thaliana and M. truncatula species. Remarkably, 7 MtbHLH and 10 MsbHLH genes were selected and validated with qRT-PCR after the treatment’s samples sampled under stressed abiotic conditions. The similar expression patterns between M. truncatula and M. sativa L have demonstrated identical expression patterns level in the root, and contrasting patterns in the stems and leaves were diverse. It was highlighted that the gene expression analyses of 17 bHLH genes were up-regulated to stresses, respectively, apart from some genes that were timely trended down-regulated to control (0 h). This study provided a concise understanding of the tissue specific of bHLH gene functions in genome-wide levels under drought, salt, and cold stresses. Our analyses provide the first insights onto the M. truncatula and M. sativa L evolution that contributes to molecular breeding for improving plant yield and stress tolerance.

中文翻译：

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Barrel Medic (Medicago truncatula) 和苜蓿 (Medicago sativa L.) 碱性螺旋-环-螺旋转录因子家族在盐和干旱胁迫下的全基因组鉴定和表达分析

基本螺旋环螺旋 (bHLH) 转录因子是植物生长和发育中最大的植物特异性转录调节因子之一，可响应生物和非生物胁迫。bHLH 的许多成员在根毛的生长和对干旱、盐分和冷胁迫的响应中发挥着重要作用。在许多物种中都发现了 bHLH 基因家族。尽管如此，到目前为止，桶医学和紫花苜蓿物种与 bHLH 的新成员仍有微小的差距。本研究旨在鉴定桶装药和苜蓿中的 bHLH 家族成员，并阐明它们的表达模式水平、网络分析、预测 3D 建模和系统发育关系。在这里，我们鉴定并表征了桶装药草和苜蓿植物中的 bHLH 基因家族及其对干旱、盐度、和冷应力。共鉴定和表征了159个MtbHLH和133个MsbHLH基因，分别分为18个亚组和17个亚组。作为一种普遍存在且流行的方法，使用了邻居加入聚类。根据系统发育分析，选择VIII和IX亚科和X亚科作为这两个物种的胁迫相关亚科。154个MtbHLH基因逐步分布在8条染色体上，23个串联重复基因，在MtbHLH家族中检测到44个重复基因片段。基因本体分析发现 bHLH 主要在这两个物种的蛋白质和 DNA 结合中起作用。Ka/Ks 的结果 < 1，这表明在拟南芥和 M. truncatula 物种之间发现了最直系同源的 bHLH 基因值。值得注意的是，在压力非生物条件下对处理样品进行采样后，选择了 7 个 MtbHLH 和 10 个 MsbHLH 基因并使用 qRT-PCR 进行了验证。M. truncatula 和 M. sativa L 之间的相似表达模式表明根中的表达模式水平相同，茎和叶中的对比模式是不同的。强调了 17 个 bHLH 基因的基因表达分析分别在胁迫下上调，除了一些及时下调至对照（0 小时）的基因。该研究提供了对干旱、盐分和冷胁迫下 bHLH 基因功能在全基因组水平上的组织特异性的简明理解。我们的分析提供了对 M. truncatula 和 M. truncatula 的初步见解。