Table_3_Comprehensive analysis of the NAC transcription factor gene family in Kandelia obovata reveals potential members related to chilling tolerance.xls

BackgroundKandelia obovata is an important mangrove species extensively distributed in Eastern Asia that is susceptible to low-temperature stress. NAC (NAM, ATAF1/2 and CUC2) domain proteins are transcription factors (TFs) that play various roles in plant growth and development and in the plant response to environmental stresses. Nevertheless, genome-wide analyses of K. obovata NAC genes (KoNACs) and their responses to chilling stress have rarely been studied.
MethodsThe KoNAC gene family was identified and characterized using bioinformatic analysis, the subcellular location of some NAC proteins was confirmed using confocal microscopy analysis, and the KoNACs that responded to chilling stress were screened using RNA-seq and qRT-PCR analysis.
ResultsA total of 79 KoNACs were identified, and they were unequally distributed across all 18 chromosomes of K. obovata. The KoNAC proteins could be divided into 16 subgroups according to the phylogenetic tree based on NAC family members of Arabidopsis thaliana. The KoNACs exhibited greater synteny with A. thaliana sequences than with Oryza sativa sequences, indicating that KoNACs underwent extensive evolution after the divergence of dicotyledons and monocotyledons. Segmental duplication was the main driving force of the expansions of KoNAC genes. Confocal microscopy analysis verified that the four randomly selected KoNACs localized to the nucleus, indicating the accuracy of the bioinformatic predictions. Tissue expression pattern analysis demonstrated that some KoNAC genes showed tissue-specific expression, suggesting that these KoNACs might be important for plant development and growth. Additionally, the expression levels of 19 KoNACs were significantly (15 positively and 4 negatively) induced by cold treatment, demonstrating that these KoNACs might play important roles during cold stress responses and might be candidate genes for the genetic engineering of K. obovata with enhanced chilling stress tolerance. Coexpression network analysis revealed that 381 coexpressed pairs (between 13 KoNACs and 284 other genes) were significantly correlated.
ConclusionsSeventy-nine KoNACs were identified in K. obovata, nineteen of which displayed chilling-induced expression patterns. These genes may serve as candidates for functional analyses of KoNACs engaged in chilling stress. Our results lay the foundation for evolutionary analyses of KoNACs and their molecular mechanisms in response to environmental stress.

背景 秋茄（Kandelia obovata）是一种广泛分布于东亚地区的重要红树林物种，且易受低温胁迫影响。NAC（NAM、ATAF1/2与CUC2）结构域蛋白属于转录因子（transcription factors, TFs），在植物生长发育以及应对环境胁迫的过程中发挥多种功能。然而，目前针对秋茄NAC基因家族（KoNACs）的全基因组分析及其对低温胁迫的响应研究尚属罕见。
方法 本研究通过生物信息学分析鉴定并注释了秋茄KoNAC基因家族；利用共聚焦显微镜分析验证了部分NAC蛋白的亚细胞定位；通过RNA-seq与qRT-PCR技术筛选出响应低温胁迫的KoNAC基因。
结果 本研究共鉴定出79个KoNAC基因，它们不均匀分布于秋茄的全部18条染色体上。基于拟南芥（Arabidopsis thaliana）NAC家族成员构建的系统发育树，可将KoNAC蛋白划分为16个亚组。相较于水稻（Oryza sativa）序列，KoNAC基因与拟南芥序列的共线性程度更高，表明KoNAC家族在双子叶与单子叶植物分化后经历了广泛的进化历程。片段重复是KoNAC基因家族扩张的主要驱动因素。共聚焦显微镜分析证实，随机选取的4个KoNAC蛋白定位于细胞核中，验证了生物信息学预测结果的准确性。组织表达模式分析显示，部分KoNAC基因具有组织特异性表达特征，提示这些基因可能在秋茄生长发育过程中发挥重要作用。此外，19个KoNAC基因的表达水平在低温处理后发生显著变化（其中15个呈上调表达，4个呈下调表达），表明这些基因可能在低温胁迫响应过程中发挥关键作用，可作为培育耐低温胁迫秋茄转基因品种的候选基因。共表达网络分析显示，13个KoNAC基因与284个其他基因之间共形成381对显著共表达关联。
结论 本研究在秋茄中共鉴定出79个KoNAC基因，其中19个呈现低温诱导表达模式。这些基因可作为研究KoNAC家族参与低温胁迫响应功能的候选对象。本研究结果为后续开展KoNAC家族的进化分析及其响应环境胁迫的分子机制研究奠定了基础。