过表达葡萄VaHY5基因增强拟南芥低温胁迫耐受性

doi:10.13560/j.cnki.biotech.bull.1985.2025-1326

摘要/Abstract

摘要：

目的探究葡萄VaHY5基因抗寒功能，为培育具有优良抗寒性状的葡萄新种质提供基因资源和理论依据。方法通过农杆菌介导的花序浸染法在拟南芥中异源过表达VaHY5基因，在-6 ℃低温胁迫下观察野生型（WT）和转基因株系的表型，测定H₂O₂和O₂·^-含量、抗氧化酶活性及膜损伤相关指标，并通过RT-qPCR检测相关抗寒基因的相对表达量变化。结果在-6 ℃低温胁迫下，过表达VaHY5株系的生长状态明显优于WT；过表达株系叶绿素荧光比WT更为明显，Fv/Fm值超过0.62，比WT高0.14以上。DAB和NBT染色结果显示，低温胁迫后3个过表达株系比WT颜色更浅，过表达植株H₂O₂和O₂·^-含量比WT低，与染色结果一致。过表达植株的可溶性糖含量及抗氧化酶活性均高于WT，但相对电导率（REL）、丙二醛（MDA）含量均低于WT，表明过表达株系细胞膜损伤程度减轻。此外，VaHY5过表达显著诱导了低温胁迫下AtCBF2（C-repeat binding factor 2）、AtCBF3（C-repeat binding factor 3）、AtCOR15a（cold-regulated 15a）和AtCOR47（cold-regulated 47）的表达，AtCBF2和AtCOR47的表达量是WT的2倍左右，AtCBF3和AtCOR15a的表达量约是WT的3‒4倍，表明VaHY5可能通过激活CBF-COR抗寒通路增强拟南芥的低温耐受性。结论过表达VaHY5通过增强活性氧（ROS）的清除能力，提高抗氧化酶的活性和可溶性糖的含量，上调冷胁迫相关基因CBFs和CORs的表达，增强转基因拟南芥对低温胁迫的耐受性。

关键词: VaHY5, 过表达, 拟南芥, 低温胁迫, 葡萄, CBF-COR通路, 抗寒性

Abstract:

Objective The aim is to explore the function of the VaHY5 gene from Vitis amurensis in response to low-temperature stress, providing genetic resources and a theoretical basis for the breeding of new grape varieties with enhanced cold resistance. Method VaHY5 was overexpressed in Arabidopsis thaliana via Agrobacterium-mediated floral dip method. Under -6 ℃ cold stress, the phenotypes of wild-type (WT) and transgenic lines were observed. The contents of H₂O₂ and O₂·^-, antioxidant enzyme activities, and membrane damage-related indicators were measured, and the relative expression levels of cold-tolerance genes were detected by RT-qPCR. Result Under low-temperature stress at -6 ℃, the growth status of the VaHY5 overexpressing line was significantly superior to that of the wild-type (WT); chlorophyll fluorescence in the overexpressing line was more pronounced than in WT, and the Fv/Fm ratio exceeded 0.62, which was 0.14 higher than the WT. DAB and NBT staining results showed that the three overexpressing lines exhibited lighter coloration than the WT after cold stress. The H₂O₂ and O₂·^- contents in the overexpressing plants were lower than those in the WT, consistent with the staining results. The soluble sugar content and antioxidant enzyme activities in the overexpressing plants were higher than those in the WT, but the relative electrical conductivity (REL) and malondialdehyde (MDA) content were lower than those in the WT. The results indicate that the degree of cell membrane damage in the overexpressing line was significantly reduced. Furthermore, VaHY5 overexpression significantly induced the expression of AtCBF2 (C-repeat binding factor 2), AtCBF3 (C-repeat binding factor 3), AtCOR15a (cold-regulated 15a), and AtCOR47 (cold-regulated 47) under low-temperature stress. The expression levels of AtCBF2 and AtCOR47 were approximately doubled compared to the wild-type (WT), while those of AtCBF3 and AtCOR15a were about 3-4 times higher than the WT. This suggests that VaHY5 may enhance cold tolerance of Arabidopsis by activating the CBF-COR cold response pathway. Conclusion Overexpression of VaHY5 enhances the scavenging of reactive oxygen species (ROS), increases the activity of antioxidant enzymes and the content of soluble sugars, and upregulates the expression of cold-stress-related genes (CBFs and CORs), thereby improving the tolerance of transgenic Arabidopsis to cold stress.

Key words: VaHY5, overexpression, Arabidopsis, low-temperature stress, grape, CBF-COR pathway, cold resistance

薛孟孟, 耿艺轩, 韩煦乐, 杨始锦. 过表达葡萄VaHY5基因增强拟南芥低温胁迫耐受性[J]. 生物技术通报, 2026, 42(10): 153-159.

XUE Meng-meng, GENG Yi-xuan, HAN Xu-le, YANG Shi-jin. Overexpression of VaHY5 Gene from Vitis amurensis Enhances Tolerance to Cold Stress in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2026, 42(10): 153-159.

图/表 5

图1 过表达VaHY5拟南芥的鉴定及VaHY5表达量检测A：PCR检测；B：拟南芥WT和转基因不同株系RT-qPCR检测；C：活体成像；M：DNA marker DL2000；Nc：阴性对照；Pc：阳性对照；WT：野生型拟南芥；OE-6-OE-15：转基因拟南芥不同株系；图中误差线表示标准误，不同小写字母表示显著性差异（P<0.05），下同

Fig. 1 Identification of Arabidopsis overexpressing VaHY5 and detection of VaHY5 relative expressionA: PCR detection. B: RT-qPCR detection of WT and transgenic Arabidopsis lines. C: Live imaging. M: DNA marker DL2000. Nc: Negative control. Pc: Positive control. WT: Wild-type Arabidopsis. OE-6-OE-15: different lines of transgenic Arabidopsis. Error bars represent standard error. Different lowercase letters indicate significant differences (P<0.05). The same below

图2 过表达VaHY5拟南芥的表型及叶绿素荧光测定A：WT和转基因株系在低温胁迫下的表型（比例尺为1 cm）；B：叶绿素荧光成像；C：Fv/Fm值

Fig. 2 Phenotype of VaHY5 overexpressing Arabidopsis and chlorophyll fluorescence measurementsA: Phenotypes of WT and transgenic lines under low-temperature stress (scale is 1 cm). B: Chlorophyll fluorescence imaging. C: Fv/Fm value

图3 组织化学染色及H2O2和O2·-含量测定

Fig. 3 Histochemical staining and determination of H2O2 and O2·- content

图4 低温胁迫下野生型和转基因拟南芥生理指标测定

Fig. 4 Measurement of physiological indicators in wild-type and transgenic Arabidopsis under cold stress

图5 低温胁迫下野生型和转基因拟南芥冷胁迫相关基因的表达量分析

Fig. 5 Expression analysis of cold stress-related genes in wild-type and transgenic Arabidopsis under low-temperature stress

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