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：

目的作为油体中含量最丰富的表面蛋白，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OLEO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 As the most abundant surface protein in oil bodies, 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 is 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, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1043.

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

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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