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林业科学 ›› 2019, Vol. 55 ›› Issue (3): 54-63.doi: 10.11707/j.1001-7488.20190306

• 论文与研究报告 • 上一篇    下一篇

刚毛柽柳NAC24基因的表达及抗逆功能分析

卢惠君, 李子义, 梁瀚予, 岳远志, 周天畅, 杨玉璋, 王玉成, 及晓宇   

  1. 东北林业大学 林木遗传育种国家重点实验室 哈尔滨 150040
  • 收稿日期:2018-04-24 修回日期:2018-06-25 出版日期:2019-03-25 发布日期:2019-04-17
  • 基金资助:
    中央高校基本科研业务费专项资金资助项目(2572017DA01);国家自然科学基金项目(31500535);东北林业大学大学生创新训练项目(201710225170)。

Expression and Stress Tolerance Analysis of NAC24 from Tamarix hispida

Lu Huijun, Li Ziyi, Liang Hanyu, Yue Yuanzhi, Zhou Tianchang, Yang Yuzhang, Wang Yucheng, Ji Xiaoyu   

  1. State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University Harbin 150040
  • Received:2018-04-24 Revised:2018-06-25 Online:2019-03-25 Published:2019-04-17

摘要: [目的] NAC类转录因子是植物特有的最大的转录因子家族之一,广泛参与植物的生长发育过程,并在植物响应盐、干旱等多种非生物胁迫的过程中发挥至关重要的调控作用。本研究拟从盐生木本植物刚毛柽柳中克隆获得一个NAC转录因子基因,研究该基因的耐盐、抗旱功能,以期为研究木本植物NAC转录因子的抗逆分子机制奠定理论基础。[方法]在刚毛柽柳NaHCO3胁迫转录组数据库中筛选获得一个NAC转录因子基因,将其命名为ThNAC24(GenBank登陆号:KF031949)。利用生物信息学工具将其与其他9个物种的NAC蛋白进行多序列比对,与拟南芥105个NAC蛋白进行进化树分析。分别用300 mmol·L-1 NaCl和400 mmol·L-1甘露醇对刚毛柽柳进行胁迫,在胁迫6、12、24和48 h后分别取刚毛柽柳根及叶组织。通过实时荧光定量RT-PCR (qRT-PCR)技术分析盐、干旱胁迫下ThNAC24基因在不同胁迫时间点及不同组织的表达情况,初步鉴定其是否响应盐、干旱胁迫。为进一步研究ThNAC24基因的抗逆功能,分别构建植物过表达(pROKⅡ-ThNAC24)及抑制表达(pFGC5941-ThNAC24)载体。利用农杆菌介导的高效瞬时遗传转化体系获得ThNAC24基因瞬时过表达(OE)、抑制表达(IE)及对照(Control)刚毛柽柳植株。在盐、干旱胁迫下分析比较了ThNAC24基因瞬时过表达、抑制表达及对照刚毛柽柳植株的二氨基联苯胺(DAB)和氯化硝基四氮唑蓝(NBT)染色情况,过氧化物酶(POD)和超氧化物歧化酶(SOD)活性,及电解质渗透率、失水率及丙二醛(MDA)含量,鉴定ThNAC24基因的耐盐、抗旱功能。[结果] ThNAC24基因的开放阅读框为1 023 bp,编码340个氨基酸。多序列比对结果显示ThNAC24在N端的氨基酸序列相似度比较高,具有NAC家族的序列特征;系统进化树分析结果显示ThNAC24与ANAC103和ANAC082的亲缘关系较近。qRT-PCR结果显示:盐胁迫下,ThNAC24基因上调表达,在根组织中胁迫12 h表达量最高,而叶组织中胁迫24 h的表达量最高;干旱胁迫下,ThNAC24基因上调表达,在根组织中胁迫6 h表达量最高,在叶组织中胁迫12 h的表达量最高。ThNAC24基因在刚毛柽柳根和叶组织中均有表达且响应盐和干旱胁迫。过表达ThNAC24基因显著降低了刚毛柽柳H2O2和超氧阴离子含量,增强了POD和SOD酶的活性,从而减少活性氧(ROS)的积累。过表达ThNAC24基因能够降低刚毛柽柳在逆境胁迫下的电解质渗透率、失水率及MDA的积累,从而保护细胞膜结构的完整性。[结论]刚毛柽柳ThNAC24基因能够响应盐、干旱胁迫,过表达ThNAC24基因植株通过增强POD和SOD活性,进而提高ROS清除能力,减少细胞受损或死亡,从而提高刚毛柽柳的耐盐及抗旱能力。

关键词: 刚毛柽柳, NAC转录因子, 基因表达, 耐盐, 抗旱

Abstract: [Objective] The NAC transcription factors are one of the largest families of plant-specific transcription factors, widely involved in plant growth and development process, and it plays a crucial role in the process responding to salt, drought and many other abiotic stresses. In present study, we cloned a NAC transcription factors gene from halophyte woody plant Tamarix hispida, to study its salt tolerance and drought resistance function in order to lay a theoretical foundation for studying the molecular mechanism of resistance to woody plant NAC transcription factors.[Method] A NAC transcription factor gene was isolated from NaHCO3 stress transcriptome database of T. hispida, and it was named as ThNAC24 (GenBank number:KF031949). Bioinformatics tools were used to perform multiple sequence alignment and phylogenetic analysis of NAC proteins from nine other species and 105 NAC proteins in Arabidopsis thaliana were analyzed. The T. hispida samples were treated with 300 mmol·L-1 NaCl and 400 mmol·L-1 Mannitol respectively, and the root and leaf tissues were taken at 6, 12, 24 and 48 h after the stress. The real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to analyze the expression of ThNAC24 gene under salt or drought stress in different time and tissues. It was initially identified whether it responded to salt or drought stress. To further study the stress resistance function of ThNAC24, overexpression (pROKⅡ-ThNAC24) and inhibitory expression vectors (pFGC5941-ThNAC24) were constructed. Overexpression (OE), inhibitory expression (IE) and control T. hispida plants were obtained by Agrobacterium-mediated high-efficient transient transformation system. The DAB, NBT staining, POD activity, SOD activity, electrolyte leakage, water loss rate, and MDA content under salt and drought stress were measured and compared with the transgenic T. hispida to identify the salt tolerance and drought resistance of the ThNAC24 gene.[Result] The open reading frame of ThNAC24 gene is 1 023 bp, encoding 340 amino acids. Multiple sequence alignments results show that the amino acid sequence of ThNAC24 is relatively high in the N-terminal and has the sequence characteristics of NAC family. Phylogenetic tree analysis showed that ThNAC24 was closely related to ANAC103 and ANAC082. The result of qRT-PCR showed that the expression of ThNAC24 gene was up-regulated under salt or drought stress. The expression level of ThNAC24 was the highest at 12 h in root tissue, while it was the highest at 24 h in leaf tissue; Under drought stress, the expression level was the highest at 6 h in root tissue, while it was the highest at 12 h in leaf tissue. The ThNAC24 gene was expressed in roots and leaves of T. hispida and responded to salt and drought stress. Overexpression of the ThNAC24 significantly reduced the contents of H2O2 and superoxide anion, enhanced the POD and SOD activities in T. hispida, thus decreased ROS accumulation. Overexpression of the ThNAC24 gene can reduce the electrolyte leakage, water loss rate and MDA accumulation of T. hispida under stress, thus protecting the integrity of cell membrane structure.[Conclusion] The ThNAC24 gene can respond to salt and drought stress. Overexpression of the ThNAC24 gene significantly enhances POD and SOD activities to improve ROS scavenging, reduces cell damage or death of T. hispida and improves its salt tolerance and drought resistance.

Key words: Tamarix hispida, NAC transcription factor, gene expression, salt tolerance, drought resistance

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