过表达砂藓RcFDH2基因提高拟南芥的耐旱性和耐高温性

doi:10.13560/j.cnki.biotech.bull.1985.2025-1056

摘要/Abstract

摘要：

目的探究砂藓（Racomitrium canescens）中甲酸脱氢酶（formate dehydrogenase, FDH）基因RcFDH2是否具有耐旱和耐高温功能，为揭示砂藓耐旱、耐高温机制以及培育抗性作物提供理论支撑。方法通过实时荧光定量PCR技术，对RcFDH2在干燥砂藓复水处理过程中的表达变化进行检测；对RcFDH2的编码序列进行克隆；获取RcFDH2过表达转基因拟南芥（Arabidopsis thaliana），并对其进行干旱和高温处理，观察植株表型并进行生理生化指标测定。结果 RcFDH2在砂藓复水过程中呈现差异表达；成功克隆获得RcFDH2的编码序列，并获取了多个RcFDH2过表达转基因拟南芥株系。在干旱及高温处理下，相比于野生型拟南芥，RcFDH2转基因株系具有更强的耐旱和耐高温能力。测定生理生化指标发现，在干旱和高温胁迫下，RcFDH2过表达转基因植株中3种渗透调节物质含量、总叶绿素含量及3种抗氧化酶活性均显著高于野生型植株，丙二醛含量低于野生型植株。结论 RcFDH2能够响应砂藓的脱水及高温胁迫处理，其过表达可能通过调节渗透调节物质和抗氧化酶活性增强拟南芥的耐旱和耐高温能力。

关键词: 砂藓, 甲酸脱氢酶基因, RcFDH2, 耐脱水性, 耐旱性, 耐高温性, 非生物胁迫

Abstract:

Objective To investigate whether a formate dehydrogenase (FDH) gene RcFDH2 of Racomitrium canescens possesses drought- and high-temperature-tolerant functions, and to provide theoretical support for revealing the drought- and high-temperature-tolerant mechanisms of R. canescens and for cultivating resistant crops. Method Real-time fluorescence quantitative PCR technology was used for the detection of the expression variations of RcFDH2 during the rehydration process of desiccated R. canescens. The coding sequence of RcFDH2 was successfully cloned. RcFDH2-overexpressed transgenic Arabidopsis thaliana lines were acquired, followed by drought and high-temperature treatments, the plant phenotypes were observed, and the physiological and biochemical indexes were determined. Result RcFDH2 showed differential expression during the rehydration process of R. canescens; the coding sequence of RcFDH2 was successfully cloned, and multiple RcFDH2-overexpressed transgenic A. thaliana lines were obtained. It was found that under drought and high-temperature treatments, the RcFDH2 transgenic lines showed stronger tolerances to drought and high-temperature compared to wild-type plants. The physiological and biochemical indicators of RcFDH2-overexpressed transgenic A. thaliana and wild-type plants revealed that in the transgenic plants, the contents of three osmotic regulatory substances, the total chlorophyll content, and the activities of three antioxidant enzymes were significantly higher than those in the wild-type plants, while the malondialdehyde content was lower than that in the wild-type plants. Conclusion RcFDH2 can respond to desiccation and high-temperature stress treatments in R. canescens, and its overexpression probably enhanced the drought and high-temperature tolerance of A. thaliana maybe by regulating osmotic regulatory substances and antioxidant enzyme activities.

Key words: Racomitrium canescens, formate dehydrogenase gene, RcFDH2, tolerance to desiccation, tolerance to drought, tolerance to high-temperature, abiotic stress

马天意, 孙铭钰, 梁舒婷, 鲍薇, 秦旭洋, 张梅娟, 沙伟, 彭疑芳. 过表达砂藓RcFDH2基因提高拟南芥的耐旱性和耐高温性[J]. 生物技术通报, 2026, 42(8): 229-238.

MA Tian-yi, SUN Ming-yu, LIANG Shu-ting, BAO Wei, QIN Xu-yang, ZHANG Mei-juan, SHA Wei, PENG Yi-fang. Overexpression of RcFDH2 from Racomitrium canescens Enhances the Tolerance of Arabidopsis thaliana to Drought and High-temperature[J]. Biotechnology Bulletin, 2026, 42(8): 229-238.

图/表 8

图1 RcFDH2在干燥砂藓复水处理过程中的表达变化误差线表示标准偏差，误差线上不同字母表示差异显著（P<0.05），下同

Fig. 1 Expression changes of RcFDH2 in desiccated R. canescens under rehydration treatmentThe error bars indicate the standard deviation, and the different letters on the error bars indicate the difference is significant (P<0.05), the same below

图2 RcFDH2的编码序列及氨基酸序列

Fig. 2 Coding sequence of RcFDH2 and its amino acid sequences

图3 RcFDH2与近缘蛋白质的序列比对红框内为FDH保守结构域，黑框内为NAD⁺结合位点，蓝色箭头处为催化位点

Fig. 3 Sequence alignment of RcFDH2 with homologous proteinsRed box indicates the conserved domains of FDH, black box indicates NAD⁺ binding sites, and blue arrow indicates the catalytic sites

图4 T2代RcFDH2过表达转基因拟南芥株系中RcFDH2的表达量分析

Fig. 4 Analysis of RcFDH2 expression in T2 generation lines of RcFDH2-overexpressed transgenic A. thaliana

图5 干旱胁迫下RcFDH2过表达转基因拟南芥的表型

Fig. 5 Phenotypesof RcFDH2-overexpressed transgenic A. thaliana under drought stress

图6 干旱胁迫下野生型及RcFDH2过表达转基因拟南芥植株的生理生化指标变化

Fig. 6 Variation in physiological and biochemical indexes of wild-type and RcFDH2-overexpressed transgenic A. thaliana under drought stress

图7 RcFDH2过表达转基因拟南芥在45 ℃高温处理下的表型

Fig. 7 Phenotypes of RcFDH2-overexpressed transgenic A. thaliana under 45 ℃ high-temperature treatments

图8 45 ℃高温处理下野生型及RcFDH2过表达转基因拟南芥的生理生化指标变化

Fig. 8 Variation in physiological and biochemical indexes of wild-type and RcFDH2-overexpressed transgenic A. thaliana under 45 ℃ high-temperature treatments

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