异源过表达桉树 EgrNAC13 提高拟南芥低温和高盐抗性

Overexpression of Eucalyptus EgrNAC13 in Arabidopsis thaliana enhances tolerance to cold and salinity

摘要

【目的】解析巨桉中 SNAC 类转录因子基因 EgrNAC13 在低温、高盐等非生物逆境抗性中的功能及其潜在调控机制，以期为利用分子辅助育种技术改良桉树抗性提供基因资源和理论基础。【方法】通过对 EgrNAC13 及其跨物种同源蛋白进行多序列比对和系统进化分析，明确其蛋白结构和进化特征。构建 35S 驱动的 EgrNAC13 过表达载体异源转化拟南芥，在分析不同株系表达量的基础上，筛选 2 个过表达株系进行低温和盐胁迫处理，观察、分析过表达株系在逆境处理下的表型。并通过 SOD、POD 活性，以及低温处理不同时间下低温响应关键基因 AtCBF1、 AtCBF2 和 AtRD29 A 的 qRT-PCR 分析，初步探究 EgrNAC13 功能发挥的潜在机制。【结果】EgrNAC13 在进化上呈现单子叶、双子叶植物，以及木本和草本植物的明显分化，其在桃金娘科植物中呈现较高的氨基酸序列相似性。不同物种 EgrNAC13 同源蛋白中，NAM 结构域序列高度相似，暗示其功能具有保守性。 EgrNAC13 过表达转基因拟南芥株系表现出明显的低温和高盐抗性。尤其是−6 ℃ 低温处理后，EgrNAC13-OE1 和 EgrNAC13-OE5 的存活率分别达到 81.6% 和 85.1%，显著高于野生型（32.3%）。4 ℃ 低温和高盐处理均可以提高过表达植株的 SOD 活性，而 POD 活性仅在高盐处理下的过表达株系中高于野生型，这表明 EgrNAC13 在低温和高盐逆境下，可以增强活性氧清除能力，提高植株的抗逆性。低温处理下，过表达植株中 AtCBF1 和 AtCBF2 的表达量分别在 4 ℃ 处理 3 h 和 12 h 显著高于野生型。而 AtRD29 A 则在正常温度处理和低温处理的不同时间都在过表达株系中被显著诱导，暗示 EgrNAC13 主要通过对 CBF 途径的调控介导了植物的低温逆境响应。【结论】在桃金娘科植物中序列高度保守的桉树 EgrNAC13 能通过提高抗氧化酶活性，增强过表达拟南芥转基因株系的低温和高盐抗性，且其逆境响应的分子调控可能主要依赖 CBF 途径。研究结果为深入解析 EgrNAC13 在桉树中的抗逆功能和调控机制奠定了研究基础，也为桉树抗逆育种提供了潜在的基因资源。

Abstract

[Objective] This study aimed to investigate the function and potential regulatory mechanisms of the EgrNAC13, a stress responsive NAC (SNAC) gene, in the abiotic stress responses of Eucalyptus grandis, particularly under cold and salinity conditions. The findings are expected to provide valuable genetic resources and a theoretical foundation for improving abiotic stress tolerance in Eucalyptus through molecular marker-assisted breeding. [Method] Multiple sequence alignment and phylogenetic analyses of EgrNAC13 and its homologs from different plant species were conducted to characterize its protein structure and evolutionary relationships. A EgrNAC13 overexpression vector, driven by 35S, was constructed and transformed into Arabidopsis thaliana. Based on transgene expression analysis of EgrNAC13 in transgenic lines, two overexpression lines were selected for cold and salt stress treatments, and their stress responsive phenotypes were evaluated. In addition, the potential regulatory mechanisms of EgrNAC13 were preliminarily explored by measuring superoxide dismutase (SOD) and peroxidase (POD) activities, as well as by analyzing the expression patterns of key cold responsive genes including AtCBF1, AtCBF2 and AtRD29 A by qRT-PCR at different time points during cold treatment. [Result] Phylogenetic analysis revealed clear evolutionary divergence of EgrNAC13 between monocotyledonous and dicotyledonous plants, as well as between woody and herbaceous species. The protein exhibited high amino acid sequence conservation among Myrtaceae species. The NAM domain sequences of EgrNAC13 homologous proteins from different species are highly similar, indicating functional conservation among these proteins. Transgenic Arabidopsis lines overexpressing EgrNAC13 displayed significantly enhanced tolerance to both cold and salinity stress. Following exposure to −6 ℃, the survival rates of EgrNAC13-OE1 and EgrNAC13-OE5 reached 81.6% and 85.1%, respectively, which were significantly higher than that of the wild type (32.3%). Under both cold and salt stress conditions, SOD activity was significantly higher in the overexpression lines than in the wild type. POD activity, however, was significantly elevated only under salt stress. This indicate that EgrNAC13 can improve plant stress tolerance to low temperature and high salt stress by enhancing the scavenging capacity of reactive oxygen species. During cold treatment, the expression levels of AtCBF1 and AtCBF2 in the overexpression lines were significantly upregulated at 3 h and 12 h of 4 ℃ treatment, respectively. In contrast, AtRD29 A expression was consistently and significantly induced in the overexpression lines under both normal and cold treatment conditions. Therefore, EgrNAC13 mediates plant responses to low temperature stress mainly by regulating the CBF pathway. [Conclusion] EgrNAC13, a highly conserved NAC transcription factor in Myrtaceae species, significantly enhances cold and salt tolerance in transgenic Arabidopsis by increasing antioxidant enzyme activities, particularly SOD and POD. Moreover, EgrNAC13 may mediate stress responses mainly through CBF-dependent pathways. These findings provide a foundation for further elucidation of the stress resistance functions and molecular mechanisms of EgrNAC13 in Eucalyptus and offer promising genetic resources for stress tolerant breeding programs.

关键词

巨桉 / NAC 转录因子 / 分子功能 / 低温 / 高盐 / 抗逆育种 / 基因表达

Key words

Eucalyptus grandis / NAC transcription factor / molecular function / low temperature / salt stress / stress-resistant breeding / gene expression

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基金资助

浙江省农业（林木）新品种选育重大科技专项(2021C02070-6)