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林业科学 ›› 2020, Vol. 56 ›› Issue (5): 69-79.doi: 10.11707/j.1001-7488.20200508

所属专题: 林木育种

• 论文与研究报告 • 上一篇    下一篇

白桦HSFA4转录因子的克隆及耐盐功能分析

刘中原,刘峥,徐颖,刘珊珊,田志兰,解庆军,高彩球*   

  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:2019-04-01 出版日期:2020-05-25 发布日期:2020-06-13
  • 通讯作者: 高彩球
  • 基金资助:
    中央高校基本科研业务费专项资金资助项目(2572018AA12);大学生创新实验训练项目(201810225473);国家自然科学基金面上项目(31670679)

Cloning and Salt Tolerance Analysis of Transcription Factor HSFA4 from Betula platyphylla

Zhongyuan Liu,Zheng Liu,Ying Xu,Shanshan Liu,Zhilan Tian,Qingjun Xie,Caiqiu Gao*   

  1. State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University Harbin 150040
  • Received:2019-04-01 Online:2020-05-25 Published:2020-06-13
  • Contact: Caiqiu Gao

摘要:

目的: 热激转录因子(HSF)在植物对非生物胁迫应答中起重要的调节作用。本研究拟从白桦中克隆获得HSFA4基因(命名为BpHSFA4),研究该基因的抗逆功能,为该基因用于林木基因工程育种奠定理论基础。方法: 在白桦转录组中筛选获得1条HSFA4基因,通过RT-PCR克隆获得BpHSFA4基因序列。利用生物信息学工具进行序列分析。利用实时荧光定量RT-PCR(qRT-PCR)分析不同逆境胁迫下BpHSFA4基因在白桦叶、茎和根中的表达模式。构建植物过表达载体pROKⅡ-BpHSFA4和抑制表达载体pFGC5941-BpHSFA4,进行白桦瞬时转化,同时以pROKⅡ瞬时侵染白桦作为对照。分析NaCl胁迫下不同瞬时转化白桦株系的MDA、H2O2、SOD、POD、相对电导率及二氨基联苯胺(DAB)、氯化硝基四氮唑蓝(NBT)及伊文思蓝(Evans blue)染色情况,以综合评定BpHSFA4基因的耐盐功能。结果: BpHSFA4基因cDNA全长为1 170 bp,编码389个氨基酸,编码蛋白的相对分子量为44.15 kDa,理论等电点为5.14,具有典型HSF结构域。非生物胁迫(NaCl、PEG6000、镉、高温和低温)条件下和激素(ABA、GA3和JA)处理下,BpHSFA4基因的表达均发生了不同程度的改变,且每种处理后至少有1个时间点发生了明显上调或者下调,其中盐胁迫下表达变化尤为明显。3种瞬时表达白桦株系中的BpHSFA4基因的表达分析结果显示成功获得了瞬时过表达转基因株系(OE)和抑制表达转基因株系(SE)。进一步对不同转基因白桦株系的生理指标和生理染色进行分析,结果显示:NaCl胁迫条件下,过表达BpHSFA4植株H2O2含量、MDA含量和相对电导率明显低于对照植株,抑制表达BpHSFA4植株的H2O2含量、MDA含量和相对电导率则明显高于对照植株;过表达BpHSFA4植株的SOD活性和POD活性明显高于对照植株,而抑制表达BpHSFA4植株的SOD活性和POD活性明显低于对照株系;NBT、DAB染色结果与H2O2含量测定结果一致,显示OE植株的着色明显比对照植株浅,而抑制表达株系着色明显比对照植株深,Evans blue染色结果和MDA含量测定结果一致。结论: BpHSFA4基因能对非生物胁迫(NaCl、PEG6000、镉、高温和低温)和激素(ABA、GA3和JA)处理做出应答,特别是盐胁迫条件下,应答强烈,该基因可能参与白桦耐盐胁迫应答。在盐胁迫条件下,瞬时过表达BpHSFA4基因株系能通过提高SOD和POD等保护酶的活性从而增强活性氧清除能力、降低膜脂氧化程度,减少细胞受损或细胞死亡,进而提高白桦的耐盐能力。本研究初步证实BpHSFA4基因是一个耐盐胁迫应答候选基因。

关键词: 白桦, HSFA4转录因子, 基因表达, 耐盐

Abstract:

Objective: Heat shock transcription factor (HSF) plays an important regulatory role in plant response to abiotic stress. The aim of this study was to identify the stress resistance function of BpHSFA4 in birch(Betula platyphylla) in order to lay a theoretical foundation for using the gene in tree breeding through genetic engineering. Methods: The BpHSFA4 cDNA sequence was obtained by searching with birch transcriptome libraries. Further, the BpHSFA4 gene was cloned by RT-PCR. And the BpHSFA4 cDNA sequence was analyzed using bioinformatics tools. Quantitative real-time RT-PCR was used to monitor the expression patterns of BpHSFA4 genes in the leaves, stems and roots of birch under different stress treatments. The vector of overexpression pROKⅡ-BpHSFA4 and silencing expression vector of pFGC5941-BpHSFA4 were constructed and transient transformed into birch. The empty pROKⅡ was also transient infected into birch as a control. The MDA content, H2O2, SOD activity, POD activity, electrolyte leakage and diaminobenzidine(DAB), nitrotetrazolium blue chloride (NBT) and Evans blue staining under NaCl stress were measured and compared between transgenic (OE, SE) and control (CK) plants. Results: The results showed that the cDNA length of BpHSFA4 was 1 170 bp. The protein encoded by this BpHSFA4 gene was 389 amino acids. The relative molecular weight of BpHSFA4 protein was 44.15 kDa, with isoelectric point 5.14. The typical HSF domain was found in the BpHSFA4 sequence.The results of qRT-PCR showed that the expressions of BpHSFA4 gene were different under abiotic stresses and hormone treatments compared to the control. Furthermore, the expressions were significantly different at least at one stress point time for each treatment. In addition, the expression patterns of BpHSFA4 gene were more significantly changed under NaCl stress. To determine whether BpHSFA4 displayed a successful overexpression(OE) or a suppressed expression (SE), the transcript levels of BpHSFA4 in the three types of transformed birch plants were measured by qRT-PCR. The results showed that the expression of BpHSFA4 was significantly higher in OE plants and clearly lower in SE plants, especially 12 h after transformation, indicating that these transient expression plants were suitable for further study. Under NaCl stress, The NBT and DAB staining and H2O2 content also showed that overexpression of BpHSFA4 markedly reduced ${\rm{O}}_2^{\bar \cdot }$ and H2O2 accumulation, while suppressed expression of BpHSFA4 led to elevated accumulation of ${\rm{O}}_2^{\bar \cdot }$ and H2O2. The SOD and POD activities of transgenic birch plants showed that OE had the highest SOD and POD activities, and that SE plants had the lowest SOD and POD activities. The Evans blue staining and MDA content and electrolyte leakage showed that cell death greatly decreased in the OE plants, but greatly increased in the SE plants. Conclusion: BpHSFA4 gene can respond to abiotic stresses and hormone treatments, and stronger under salt stress, it indicates that the gene may be involved in the response to salt stress in birch trees. All results indicated that overexpression of BpHSFA4 improved the salt stress tolerance by increasing the ability to scavenge reactive oxygen species (ROS), the activities of SOD and POD to reduce the accumulation of H2O2 and ${\rm{O}}_2^{\bar \cdot }$, thereby reducing cell damage or cell death and enhancing birch resistance. BpHSFA4 was proved to be a candidate gene responding to stresses.

Key words: Betula platyphylla, HSFA4 transcription factor, gene expression, salt tolerance

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