过表达砂藓RcELIP1提高转基因拟南芥耐旱性及耐高温性

doi:10.13560/j.cnki.biotech.bull.1985.2025-1065

摘要/Abstract

摘要：

目的探究砂藓（Racomitrium canescens）中早期光诱导蛋白（early light-induced proteins, ELIPs）基因RcELIP1在植物抗旱、抗高温胁迫中的功能并探究其抗逆机制。方法利用实时荧光定量PCR检测RcELIP1在砂藓脱水、复水及45 ℃高温处理中的表达变化，通过生物信息学方法预测其编码蛋白质特性，克隆RcELIP1的编码序列构建过表达载体并将其转化到拟南芥（Arabidopsis thaliana）中，获取RcELIP1过表达转基因拟南芥株系，对比转基因株系与野生型拟南芥在干旱、高温胁迫下的表型差异，并测定胁迫下各项生理生化指标。结果实时荧光定量PCR结果证实RcELIP1在砂藓脱水、高温胁迫中显著差异表达。RcELIP1含216个氨基酸残基，预测具有叶绿素a/b结合域，为亲水性不稳定跨膜蛋白，定位于叶绿体。RcELIP1过表达转基因拟南芥在干旱、高温下存活率显著高于野生型植株，转基因株系能维持更高的叶绿素和脯氨酸含量及抗氧化酶活性，以及较低的丙二醛水平。结论过量表达的RcELIP1可能通过保护叶绿体功能、增强渗透调节以及抗氧化能力，提高植物对干旱和高温胁迫的适应能力。

关键词: 砂藓, 早期光诱导蛋白基因, RcELIP1, 耐旱性, 耐高温性

Abstract:

Objective This study investigated the function of the early light-induced protein (ELIP) gene RcELIP1 from the moss Racomitrium canescens in conferring tolerance to drought and high-temperature stress, and also preliminarily explored the underlying mechanisms, aiming to provide further theoretical insight into the role of bryophyte ELIPs in plant stress responses. Method The expression patterns of RcELIP1 in R. canescens during dehydration, rehydration, and 45 ℃ high-temperature stress were analyzed using quantitative real-time PCR (RT-qPCR), the characteristics of the encoded protein were predicted via bioinformatics tools, the coding sequence of RcELIP1 was cloned into an overexpression vector and transformed into Arabidopsis thaliana to generate transgenic lines, phenotypic differences between transgenic and wild-type plants under drought and high-temperature stress were compared, and physiological and biochemical indices were measured. Result RT-qPCR analysis confirmed that RcELIP1 expression was significantly altered in response to both dehydration and high-temperature stress. RcELIP1 comprises 216 amino acid residues, it was predicted to contain a chlorophyll a/b-binding domain, and was characterized as a hydrophilic, unstable transmembrane protein localized to chloroplasts. Under drought and high-temperature stress, transgenic RcELIP1-overexpressed A. thaliana showed significantly higher survival rates than wild-type plants, the transgenic lines also maintained higher chlorophyll and proline content, elevated antioxidant enzyme activities, and lower malondialdehyde levels. Conclusion The overexpression of RcELIP1 enhances plant tolerance to drought and high-temperature stress, likely through mechanisms involving chloroplast protection, increased osmotic regulation, and improved antioxidant capacity.

Key words: Racomitrium canescens, early light-induced protein gene, RcELIP1, tolerance to drought, tolerance to high-temperature

曹婉笛, 鲍薇, 孙天国, 梁舒婷, 秦旭洋, 张梅娟, 沙伟, 彭疑芳, 马天意. 过表达砂藓RcELIP1提高转基因拟南芥耐旱性及耐高温性[J]. 生物技术通报, 2026, 42(7): 246-256.

CAO Wan-di, BAO Wei, SUN Tian-guo, LIANG Shu-ting, QIN Xu-yang, ZHANG Mei-juan, SHA Wei, PENG Yi-fang, MA Tian-yi. Overexpression of RcELIP1 from Racomitrium canescens Enhances the Tolerance to Drought and High Temperature in Transgenic Arabidopsisthaliana[J]. Biotechnology Bulletin, 2026, 42(7): 246-256.

图/表 8

图1 RcELIP1在砂藓复水、脱水及高温处理过程中的表达变化分析A：干燥砂藓复水处理；B：脱水处理；C：45 ℃高温胁迫处理。误差线表示标准偏差，不同字母表示差异显著（P<0.05），下同

Fig. 1 Analysis of RcELIP1 expression changes in Racomitrium canescens during rehydration, dehydration, and high-temperature treatmentsA: Desiccated R. canescens under rehydration treatments. B: Dehydration treatments. C: 45 ℃ high-temperature treatments. The error bars indicate the standard deviation, the different letters indicate that the difference is significant (P<0.05), the same below

图2 RcELIP1编码序列的克隆及序列信息A：RcELIP1编码序列的PCR扩增结果（M：DL 2000 DNA marker；-：水对照；1，2：RcELIP1编码序列）；B：RcELIP1的核苷酸序列与编码的氨基酸序列

Fig. 2 Cloning of the coding sequence and the sequence information of RcELIP1A: PCR amplification results of RcELIP1 coding sequence (M: DL 2000 DNA marker; -: water control; 1,2: RcELIP1 coding sequence). B: The nucleotide sequence and the encoded amino acid sequence of RcELIP1

图3 RcELIP1的生物信息学预测分析A. 亲/疏水性预测；B. 跨膜结构预测；C. 蛋白质二级结构预测，蓝色：α螺旋，绿色：β折叠，红色：无规则卷曲，紫色：延伸链；D. 蛋白质三级结构预测；E. 信号肽预测；F. RcELIP1与同源蛋白质的序列比对

Fig. 3 Bioinformatics prediction analysis of RcELIP1A. Prediction of hydrophilicity and hydrophobicity. B. Transmembrane structure prediction. C. Protein secondary structure prediction; blue: alpha helix, green: beta turn, red: random coil, purple: extended strand. D. Protein tertiary structure prediction. E. Signal peptide prediction. F. Sequence alignment of RcELIP1 and homologous proteins

图4 RcELIP1过表达转基因拟南芥植株的筛选及鉴定A：T1代RcELIP1过表达转基因拟南芥植株的抗生素筛选，红圈内为成功筛选出的T1代植株；B：RcELIP1过表达转基因拟南芥T2代植株中RcELIP1的表达量分析

Fig. 4 Screening and identification of transgenic RcELIP1-overexpressed transgenic A.thaliana plantsA: Antibiotic screening of RcELIP1 overexpressed transgenic Arabidopsis thaliana plants, the red circles indicate the successfully screened RcELIP1 T1 generation plants; B: Expression analysis of RcELIP1 in T2 generation RcELIP1-overexpressedtransgenic A. thaliana plants

图5 干旱胁迫下RcELIP1过表达转基因拟南芥植株的表型

Fig. 5 Phenotypes performance of RcELIP1-overexpressed transgenic A.thaliana plants under drought stress

图6 干旱胁迫下野生型及RcELIP1过表达转基因拟南芥植株生理生化指标的变化

Fig. 6 Changes in physiological and biochemical indexes of wild-type and RcELIP1-overexpressed transgenic A. thaliana under drought stress

图7 45 ℃高温处理下RcELIP1过表达转基因拟南芥的表型

Fig. 7 Phenotypes of RcELIP1-overexpressed transgenic A. thaliana under 45 ℃ high-temperature treatments

图8 45 ℃高温处理下野生型及RcELIP1过表达转基因拟南芥生理生化指标的变化

Fig. 8 Changes in physiological and biochemical indexes of wild-type and RcELIP1-overexpressed transgenic A. thaliana under 45 ℃ high-temperature treatments

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