水稻热激转录因子HsfA2b调控非生物胁迫抗性的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0908

摘要/Abstract

摘要：

【目的】水稻在生长发育过程中，常会受到各种非生物胁迫而严重影响产量。热激转录因子作为植物抗逆过程中的一个重要元件，通过调控一系列胁迫响应基因的表达以提高植物抗逆性。探究水稻热激转录因子OsHsfA2b调控非生物胁迫的功能与初步机理，为培育水稻抗逆新品种提供了优异基因资源和理论支撑。【方法】通过构建OsHsfA2b过量表达和RNAi的转基因水稻，分别观察水稻幼苗在高温、低温、干旱和高盐处理后的抗逆表型，统计存活率。同时，在逆境胁迫处理后，通过DAB染色检测水稻叶片活性氧（ROS）含量及RT-qPCR检测抗氧化途径相关基因OsSOD和OsCAT的表达量，分析OsHsfA2b对抗氧化途径的调控作用。【结果】在高温、低温、干旱和高盐等非生物胁迫条件下，水稻热激转录因子OsHsfA2b被显著诱导表达。与野生型NPB相比，OsHsfA2b过量表达转基因水稻对非生物胁迫的抗性明显增强，植株损伤程度较轻，存活率提高。相反，OsHsfA2b-RNAi水稻对非生物胁迫更加敏感，植株损伤严重，存活率降低。此外，在非生物胁迫条件下，OsHsfA2b过量表达转基因水稻比NPB和OsHsfA2b-RNAi水稻体内活性氧的含量减少，并且OsHsfA2b显著诱导了抗氧化途径相关基因OsSOD和OsCAT的表达，OsHsfA2b参与抑制水稻体内活性氧的积累以降低逆境胁迫诱导的活性氧大量积累对植株的伤害。【结论】非生物胁迫下，水稻热激转录因子OsHsfA2b被显著诱导表达，可通过抗氧化途径正调控水稻对逆境胁迫的抗性。

关键词: 水稻, HsfA2b, 热激转录因子, 非生物胁迫, 抗性, 活性氧

Abstract:

【Objective】 Rice is often affected by various abiotic stresses during growth and development, which severely restricts rice yield. Heat shock transcription factors（HSFs）, as an important element of resistance to abiotic stresses in plant, can enhance plant resistance to abiotic stresses by regulating the expressions of a series of stress related genes. This study aims to investigate the function and mechanism of rice heat shock transcription factor OsHsfA2b regulating abiotic stresses, which may provide excellent gene resource and theoretical support for cultivating new rice varieties with stress resistance.【Method】 We constructed OsHsfA2b-overexpressed and RNAi rice transgenic plants, then their seedlings were treated with high temperature, low temperature, drought, and high salt, respectively, and their phenotypes were observed and survival rates were counted. The detection of reactive oxygen species（ROS）deposition by DAB staining, and expression levels of antioxidant pathway related genes OsSOD and OsCAT with RT-qPCR after abiotic stress treatment, aiming to analyze the regulatory effect of OsHsfA2b on the antioxidant pathway.【Result】 The rice HSF gene OsHsfA2b was induced significantly by abiotic stress conditions, such as high temperature, low temperature, drought, and high salt. Compared with the wild-type plants, OsHsfA2b-overexpressed transgenic plants significantly enhanced the resistance to abiotic stresses, as well as survival rate, and less damage. On the contrary, both resistance and survival rate of OsHsfA2b-RNAi plants to abiotic stresses decreased, and the plants were severely damaged. Moreover, the ROS deposition in OsHsfA2b-overexpressed plants decreased compared with NPB and RNAi plants under abiotic stress. Correspondingly, OsHsfA2b induced the expressions of antioxidant pathway related genes OsSOD and OsCAT, suggesting that OsHsfA2b suppressed ROS accumulation to reduce its damage caused by abiotic stress induction.【Conclusion】 The above results indicate that OsHsfA2b is induced by abiotic stresses and positively regulates rice resistance to abiotic stresses through antioxidant pathways, and it is an excellent gene resource for rice stress-resistance breeding.

Key words: Oryza sativa L., HsfA2b, heat shock transcription factor, abiotic stress, resistance, reactive oxygen species

邹修为, 岳佳妮, 李志宇, 戴良英, 李魏. 水稻热激转录因子HsfA2b调控非生物胁迫抗性的功能分析[J]. 生物技术通报, 2024, 40(2): 90-98.

ZOU Xiu-wei, YUE Jia-ni, LI Zhi-yu, DAI Liang-ying, LI Wei. Functional Analysis of Rice Heat Shock Transcription Factor HsfA2b Regulating the Resistance to Abiotic Stresses[J]. Biotechnology Bulletin, 2024, 40(2): 90-98.

图/表 8

表1 实时荧光定量PCR引物

Table 1 Primers for RT-qPCR

引物Primer	序列Sequence（5'-3'）
UBQ-F	AACCAGCTGAGGCCCAAGA
UBQ-R	ACGATTGATTTAACCAGTCCATGA
OsHsfA2b-F	GCCTTGTTTGATCCCCAGGA
OsHsfA2b-R	TTGTCGTTCAACTCCCCCTG
OsSOD-F	AACAAAGGCAGGGCTGTAGA
OsSOD-R	TAAGGTTCCAGGGCATCAAG
OsCAT-F	TGAAGCCAAGCATGTGAAGA
OsCAT-R	GCCCAACGACAACAGAAGAT

图1 OsHsfA2b的CDS扩增电泳图 M：Marker；N：阴性对照；1：OsHsfA2b CDS全长扩增片段

Fig. 1 Electropherogram of OsHsfA2b gene CDS amplification M: Marker; N: negative control; 1: OsHsfA2b full-length CDS amplified fragment

图2 OsHsfA2b的蛋白结构结构图上方数字表示氨基酸位置。DBD：DNA结合结构域；OD：寡聚化结构域；NLS：核定位信号；CTAD：C端转录激活结构域；NES：C端核输出信号

Fig. 2 Protein structure of OsHsfA2b The number above the structural diagram represents the position of amino acids. DBD: DNA binding domain; OD: oligomerization domain; NLS: nuclear localization signal; CTAD: C-terminal transcriptional activation domain; NES: nuclear export signal

图3 OsHsfA2b-OX和OsHsfA2b-RNAi转基因植株的鉴定 A：OsHsfA2b-OX转基因植株中OsHsfA2b的表达情况；B：OsHsfA2b-RNAi植株中OsHsfA2b的表达情况

Fig. 3 Identification of OsHsfA2b-OX and OsHsfA2b-RNAi transgenic plants A: OsHsfA2b gene expression of OsHsfA2b-OX transgenic plants. B: OsHsfA2b gene expression of OsHsfA2b-RNAi transgenic plants

图4 高温胁迫下转基因植株的表型和OsHsfA2b的表达 A ：高温胁迫（42℃）NPB 植株中OsHsfA2b 的表达量；B ：高温处理后OsSOD 的表达；C ：高温处理2 d 后植株的表型; D : 存活率统计；E：高温处理后OsCAT 的表达；F ：活性氧含量的检测。* 表示差异显著（P<0.05），** 表示差异极显著（P<0.01），下同

Fig. 4 Phenotypes and OsHsfA2b expressions of transgenic plants under high temperature stress A: Expression of OsHsfA2b in NPB under high temperature stress（42℃）. B: Expression of OsSOD after high temperature treatment. C: Phenotypes of plants after high temperature treatment for 2 d after high temperature treatment. D: Survival rate statistic. E: Expression of OsCAT after high temperature treatment. F: Determination of reactive oxygen species content. * indicates significant difference（P<0.05）, and **indicates extremely significant difference（P<0.01）. The same below

图5 低温胁迫下转基因植株的表型和OsHsfA2b的表达 A：低温胁迫（4℃）OsHsfA2b的表达量；B：低温处理2 d后的表型；C：存活率统计；D-E：低温处理后OsSOD和OsCAT的表达量

Fig. 5 Phenotypes and OsHsfA2b expressions of transgenic plants under low temperature stress A: Expression of OsHsfA2b under low temperature stress（4℃）. B: Phenotypes of plants after low temperature treatment for 2 d. C: Survival rate statistic. D-E: Expression of OsSOD and OsCAT after low temperature treatment

图6 干旱胁迫下转基因植株的表型和OsHsfA2b的表达 A：干旱处理OsHsfA2b的表达量；B：干旱处理1 d后的表型；C：存活率统计；D-E：干旱处理后OsSOD和OsCAT的表达量

Fig. 6 Phenotypes and OsHsfA2b expressions of transgenic plants under drought stress A: Expression of OsHsfA2b under drought treatment. B: Phenotypes of plants after drought treatment for 1 d. C: Survival rate statistic. D-E: Expression of OsSOD and OsCAT after drought treatment

图7 高盐胁迫下转基因植株的表型和OsHsfA2b的表达 A：150 mmol/L NaCl处理后OsHsfA2b的表达量；B：150 mmol/L NaCl处理后种子萌发5 d的表型；C：150 mmol/L NaCl处理2 d后的表型；D-E：高盐处理后OsSOD和OsCAT的表达量

Fig. 7 Phenotypes and OsHsfA2b expressions of transgenic plants under high salt stress A: Expression of OsHsfA2b under 150 mmol/L NaCl. B: Phenotypes of seedlings geminated after 150 mmol/L NaCl for 5 d. C: Phenotypes of plants under 150 mmol/L NaCl for 2 d. D-E: Expression of OsSOD and OsCAT after high salt treatment

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