VvHSP18.2过表达调节葡萄盐碱抗性的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0692

摘要/Abstract

摘要：

目的盐碱胁迫严重影响葡萄生长、发育和品质形成，评价VvHSP18.2在调节葡萄盐碱抗性上的功能，鉴定葡萄抗盐碱关键基因，为葡萄抗逆遗传改良奠定理论基础。方法运用生物信息学技术构建VvHSP18.2进化树，分析其启动子，利用RT-qPCR技术分析VvHSP18.2在NaHCO₃等各种胁迫处理后的表达水平，将VvHSP18.2在拟南芥和葡萄中过表达，通过表型分析和生理指标鉴定其功能。结果 VvHSP18.2属于热激蛋白CI亚家族，主要在葡萄愈伤组织、果实和根系中表达。其启动子区含有多个响应干旱、盐、高温胁迫的顺式作用元件。VvHSP18.2快速响应多个非生物胁迫；在处理后6和12 h，VvHSP18.2的表达受NaCl，尤其是NaHCO₃显著诱导；在处理后1 h，其表达受PEG6000显著诱导；在AlCl₃处理后3和6 h，VvHSP18.2的表达显著降低。在葡萄根系和拟南芥中，VvHSP18.2过表达能缓解NaHCO₃处理导致的表型伤害，降低电导率和MDA含量，提高NaHCO₃胁迫抗性。并且，VvHSP18.2过表达提高了拟南芥SOD、CAT、POD酶活性，降低O^2-和H₂O₂积累水平，表明其通过提高活性氧清除能力来提高胁迫抗性。相比之下，VvHSP18.2过表达对拟南芥NaCl抗性无显著影响。结论 VvHSP18.2参与调控葡萄胁迫应激反应，VvHSP18.2过表达通过提高植物抗氧化酶活性和活性氧清除能力来调控活性氧代谢平衡，进而提高葡萄和拟南芥的NaHCO₃胁迫抗性。

关键词: 葡萄, VvHSP18.2, 碱性盐胁迫, 活性氧, 拟南芥

Abstract:

Objective Saline-alkali stress severely affects grape growth, development, and quality formation. This study aims to evaluate the function of VvHSP18.2 in regulating grape saline-alkali resistance, identify key genes involved in grape salt-alkali tolerance, and establish a theoretical foundation for the genetic improvement of stress resistance in grapes. Method Phylogenetic analysis of VvHSP18.2 was conducted using bioinformatics tools, and its promoter was analyzed. RT-qPCR was used to assess the expression of VvHSP18.2 under various stress treatments, including NaHCO₃. VvHSP18.2 was overexpressed in Arabidopsis and grape, and its function was evaluated through phenotypic observation and physiological indicators. Result VvHSP18.2 belonged to the heat shock protein CI subfamily and is mainly expressed in grape callus, fruit, and roots. Its promoter region contained multiple cis-acting elements responsive to drought, salt, and high-temperature stresses. VvHSP18.2 rapidly responded to multiple abiotic stresses: its expression was significantly induced by NaCl, especially NaHCO₃, at 6 and 12 h post-treatment; it was significantly induced by PEG6000 at 1 hour post-treatment; and its expression significantly decreased at 3 and 6 h after AlCl₃ treatment. The overexpression of VvHSP18.2 in grape roots and Arabidopsis alleviated phenotypic damage caused by NaHCO₃ treatment, reduced electrolyte leakage and MDA content, and enhanced resistance to NaHCO₃ stress. Furthermore, VvHSP18.2 overexpression increased the activities of SOD, CAT, and POD enzymes in Arabidopsis, reduced the accumulation of O^2- and H₂O₂, indicating that it enhanced stress resistance by improving reactive oxygen species (ROS) scavenging capacity. In contrast, VvHSP18.2 overexpression had no significant effect on NaCl resistance in Arabidopsis. Conclusion VvHSP18.2 is involved in regulating the stress response in grapes. The overexpression of VvHSP18.2 enhances saline-alkali stress resistance in both grape and Arabidopsis by increasing antioxidant enzyme activities and improving ROS scavenging capacity, thereby regulating the balance of ROS metabolism.

Key words: grapevines, VvHSP18.2, alkaline salt stress, reactive oxygen species, Arabidopsis thaliana

徐玉娇, 孙玉帅, 刘道奇, 张丽, 张志昌, 姚玉新. VvHSP18.2过表达调节葡萄盐碱抗性的功能分析[J]. 生物技术通报, 2026, 42(4): 161-169.

XU Yu-jiao, SUN Yu-shuai, LIU Dao-qi, ZHANG Li, ZHANG Zhi-chang, YAO Yu-xin. Functional Analysis of VvHSP18.2 Overexpression in Regulating Salt-alkali Resistance in Grapevines[J]. Biotechnology Bulletin, 2026, 42(4): 161-169.

图/表 6

表1 RT-qPCR引物序列

Table 1 Primer sequences for RT-qPCR

引物

Primer

正向引物

Forward primer （5′‒3′）

反向引物

Reverse primer （5′‒3′）

GAGATTCCGTTGTCCAGAAGTC

CAATGTTGCCATAGAGGTCCTT

图1 VvHSP18.2系统进化及表达分析A：拟南芥和葡萄HSP蛋白的系统进化树；B：VvHSP18.2上游2 000 bp启动子分析

Fig. 1 Phylogenetic evolution and expression analysis of VvHSP18.2A: Phylogenetic tree of HSP proteins in Arabidopsis thaliana (At) and Vitis vinifera (Vv). B: Analysis of the 2 000 bp promoter before VvHSP18.2

图2 VvHSP18.2组织特异性分析和非生物胁迫响应分析A：VvHSP18.2组织特异性分析；B：NaHCO3处理；C：NaCl处理；D：AlCl3处理；E：PEG6000处理。不同的小写字母表示显著差异（P<0.05）。下同

Fig. 2 VvHSP18.2 tissue-specific analysis and abiotic stress response analysisA: VvHSP18.2 tissue-specific analysis. B: NaHCO3 treatment. C: NaCl treatment. D: AlCl3 treatment. E: PEG6000treatment. Different lowercase letters indicate significant differences (P<0.05). The same below

图3 过表达VvHSP18.2减轻葡萄根系碱性盐胁迫伤害A：VvHSP18.2转基因葡萄根系鉴定（NC：阴性对照）；B：转基因葡萄根系受碱性盐胁迫伤害程度表型；C：转基因葡萄根系中VvHSP18.2的表达水平；D：MDA含量；E：根系活力

Fig. 3 Overexpression of VvHSP18.2 alleviates alkaline salt stress injury in grapevine rootsA: Root system identificationof VvHSP18.2 transgenic grape (NC: Negative control). B: Phenotype of damage to the root system of genetically modified grapes under alkaline salt stress. C: The expressions of VvHSP18.2 in the root system of genetically modified grapes. D: MDA content. E: Root activity

图4 VvHSP18.2转基因拟南芥的鉴定和过表达VvHSP18.2增强拟南芥NaHCO3抗性A：VvHSP18.2转基因拟南芥鉴定；B：转基因拟南芥中VvHSP18.2的表达水平；C：表型；D：根系活力；E：MDA含量；F：相对电导率；G：SOD活性；H：CAT活性；I：POD活性；J：NBT染色（左）和DAB染色（右）；WT：野生型拟南芥；L1、L2、L3：3个VvHSP18.2过表达拟南芥株系

Fig. 4 Identification and overexpression of VvHSP18.2 transgenic Arabidopsis thaliana to enhance Arabidopsis NaHCO3 toleranceA: Identification of VvHSP18.2 transgenic Arabidopsis. B: Expressions of VvHSP18.2 in transgenic Arabidopsis phenotype chart. C: Phenotype chart. D: Root activity content. E: MDA content. F: Relative electrical conductivity. G: SOD activity. H: CAT activity. I: POD activity. J: NBT staining (left) and DAB staining (right). WT: Wild-type Arabidopsisthaliana background. L1, L2, and L3: Three transgenic lines harboring the VvHSP18.2 overexpression construct

图5 过表达VvHSP18.2对拟南芥NaCl抗性的影响A：表型；B：鲜重；C：根长；D：侧根数目

Fig. 5 Effect of overexpression of VvHSP18.2 on NaCl resistance in Arabidopsis thalianaA: Phenotypes. B: Fresh weight. C: Root length. D: Number of lateral roots

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