Overexpression of VaHY5 Gene from Vitis amurensis Enhances Tolerance to Cold Stress in Arabidopsis thaliana

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

Abstract

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

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

Figures/Tables 5

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

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

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

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

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

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