过表达砂藓RcOLEO1基因增强拟南芥的耐旱性及耐高温性

doi:10.13560/j.cnki.biotech.bull.1985.2025-1043

摘要/Abstract

摘要：

目的油体蛋白（oleosin, OLEO, OLE）对植物油体形成及油脂储存至关重要，同时在植物应对逆境胁迫过程中也发挥重要作用。砂藓（Racomitrium canescens）是一种拥有强耐脱水性和耐高温性的典型旱生苔藓植物，通过验证砂藓中响应胁迫处理的油体蛋白基因RcOLEO1能否提高植物抗逆性，为进一步阐明砂藓耐旱及耐高温过程的分子机制，以及挖掘砂藓相关抗旱基因资源提供理论依据。方法利用实时荧光定量PCR（real-time quantitative PCR, RT-qPCR）检测复水、脱水和高温胁迫处理下砂藓中RcOLEO1的表达模式，对RcOLEO1的编码序列进行克隆并构建其过表达拟南芥（Arabidopsis thaliana）株系，对转基因拟南芥分别进行干旱及高温胁迫处理，观察表型，并对生理生化指标进行测定。结果 RT-qPCR结果显示RcOLEO1响应砂藓的复水、脱水和高温胁迫处理；成功克隆得到RcOLEO1的编码序列，获得T₂代过表达转基因拟南芥株系；对野生型及过表达转基因拟南芥进行高温及干旱胁迫处理，发现过表达砂藓RcOLEO1增强了拟南芥的耐旱性及耐高温性；对渗透调节物质含量、丙二醛含量、叶绿素含量等生理生化指标进行检测，初步阐释了过表达RcOLEO1增强拟南芥植株的耐旱性及耐高温性的生理响应方式。结论过表达RcOLEO1可能通过调控渗透调节物质积累与抗氧化酶活性，增强植物对干旱和高温胁迫的耐受性。

关键词: 砂藓, RcOLEO1, 油体蛋白基因, 干旱胁迫耐性, 高温胁迫耐性, 基因表达, 生物信息学分析, 生理生化指标检测

Abstract:

Objective Oleosin (OLEO, OLE) play crucial roles in oil body formation and lipid storage in plants, while also contributing significantly to the stress resisting process of plants. Racomitrium canescens is a typical xerophytic moss exhibiting extreme tolerance to desiccation and high-temperature. This study aimed to investigate whether the stress-responsive oleosin gene RcOLEO1 from R. canescens can enhance plant resistance to stress, providing a theoretical foundation for elucidating the molecular mechanisms of underlying drought and high-temperature in R. canescens and identifying potential drought-resistant genetic resources. Method The expression patterns of RcOLEO1 under rehydration, dehydration and high-temperature stress treatments were analyzed using real-time quantitative PCR (RT-qPCR). The coding sequence of RcOLEO1 was cloned and used to construct overexpressing A. thaliana lines, the transgenic A. thaliana plants were subjected to have drought and high-temperature stress treatments, respectively, for phenotypic observations and physiological and biochemical indexes measurements. Result RT-qPCR results revealed that RcOLEO1 was responsive to rehydration, dehydration and high-temperature stress treatments in R. canescens. The coding sequence of RcOLEO1 was successfully cloned, and T₂ generation transgenic A. thaliana overexpressing RcOLEO1 was obtained. Under drought and high-temperature stress, RcOLEO1-overexpresion lines demonstrated enhanced tolerance compared to wild-type plants. Physiological analyses, including measurements of osmoregulatory substances content, malondialdehyde content, and chlorophyll content, preliminarily elucidated the mechanisms by which the overexpression of RcOLEO1 improved drought and high-temperature tolerance in A. thaliana. Conclusion The overexpression of RcOLEO1 likely enhances plant tolerance to drought and high-temperature stress by modulating the accumulation of osmoregulatory compounds and antioxidant enzyme activity.

Key words: Racomitrium canescens, RcOLEO1, oleosin gene, tolerance to drought stress, tolerance to high-temperature stress, gene expression, bioinformatics analysis, detection of physiological and biochemical indexes

郭苗, 许家佳, 孙天国, 蔡璨, 曹婉笛, 包纪星, 沙伟, 张梅娟, 彭疑芳, 马天意. 过表达砂藓RcOLEO1基因增强拟南芥的耐旱性及耐高温性[J]. 生物技术通报, 2026, 42(5): 312-322.

GUO Miao, XU Jia-jia, SUN Tian-guo, CAI Can, CAO Wan-di, BAO Ji-xing, SHA Wei, ZHANG Mei-juan, PENG Yi-fang, MA Tian-yi. Overexpression of RcOLEO1 Enhanced the Tolerance of Arabidopsis thaliana toDrought and High-temperature Stress[J]. Biotechnology Bulletin, 2026, 42(5): 312-322.

图/表 7

图1 砂藓复水及脱水处理过程中RcOLEO1的表达分析误差线表示标准偏差，误差线上字母表示不同的差异显著性（P<0.05），下同

Fig. 1 Expression analysis of RcOLEO1 during rehydration and dehydration treatments of Racomitrium canescensThe error bars indicates the standard deviation, and the letters on the error bars indicate different significance (P<0.05), the same below

图2 RcOLEO1与近缘蛋白质的进化关系分析（A）及序列比对（B）红框内为脯氨酸结motif

Fig. 2 Phylogenic analysis (A) and sequence alignment (B) of RcOLEO1 and homologous proteinsThe red boxes indicate the conserved proline knot motif

图3 RcOLEO1过表达转基因拟南芥的筛选及鉴定A：RcOLEO1过表达转基因拟南芥T1代植株的筛选；B：RcOLEO1过表达转基因拟南芥T2代植株的鉴定

Fig. 3 Screening and characterization of RcOLEO1 overexpression transgenic A. thalianaA: Screening of T1 generation plants of transgenic A. thaliana overexpressing RcOLEO1.B: Identification of T2RcOLEO1 overexpression transgenic A. thaliana

图4 干旱胁迫下RcOLEO1过表达转基因拟南芥植株的表型

Fig. 4 Phenotypes of RcOLEO1 overexpression transgenic A. thaliana under drought stress

图5 干旱处理下野生型和RcOLEO1过表达转基因拟南芥植株生理生化指标的变化

Fig. 5 Changes in physiological and biochemical indexes of wild-type and RcOLEO1 overexpression transgenic A. thaliana under drought treatment

图6 45 ℃高温处理下RcOLEO1过表达转基因拟南芥植株表型

Fig. 6 Phenotypes of RcOLEO1 overexpression transgenic A. thaliana plants under 45 ℃ high-temperature treatment

图7 45 ℃高温处理下野生型和RcOLEO1过表达转基因拟南芥植株生理生化指标变化

Fig. 7 Changes in physiological and biochemical indexes of wild-type and RcOLEO1 overexpression transgenic A. thaliana under 45 °C high-temperature treatment

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