葡萄VvGAI1与VvJAZ9蛋白互作及低温下的表达模式分析

doi:10.3864/j.issn.0578-1752.2023.15.012

Abstract

Abstract:

【Objective】 DELLA protein belongs to plant-specific GRAS protein family, which is a significant regulatory factor in GA signal transduction pathway and plays important roles in plant growth, development and resistance to different stresses. In this study, the European grapevine VvGAI1 gene was cloned and the analysis of subcellular localization, protein interaction and expression were performed, so as to lay the foundation for the further study of the function of DELLA protein in response to cold stress in grapevine.【Method】 The VvGAI1 gene sequence was obtained by homologous cloning from the leaves of Vitis vinifera cv. Chardonnay. The VvGAI1 sequence was analyzed by bioinformatics method, and the multiple sequence alignment and phylogenetic trees were performed by DNAMAN and MEGA7.0, respectively. The location of VvGAI1 protein in cells was determined by subcellular localization, and the transcriptional activation activity of the VvGAI1 protein was confirmed by a yeast assay. The interaction between VvGAI1 protein and VvJAZ9 protein was verified by yeast two-hybrid and BiFC assays. The VvGAI1 protein polyclonal antibodies from rabbit was prepared by VvGAI1 protein purified from prokaryotic expression. The VvGAI1 protein expression at low temperature was detected by Western blot method. The effect of exogenous MeJA and GA₃ on the cold resistance in grape was analyzed by relative electrical conductivity.【Result】 The VvGAI1 gene was cloned from Chardonnay leaves, with carrying an ORF of 1 773 bp, encoding 590 amino acids, locating on chromosome 1, and containing only one exon. The VvGAI1 protein had a molecular weight of 64.87 kDa and pI of 5.31, which was an acidic unstable hydrophilic protein. The VvGAI1 belonged to GRAS family and had the conserved DELLA and GRAS domains. Protein clustering analysis showed that VvGAI1 was closely related to Arabidopsis GAI and tobacco GAI1. The results of subcellular location and transcriptional activation showed that VvGAI1 was a transcription factor localized in the nucleus and had transcriptional self-activation activity. The interaction between VvGAI1 and VvJAZ9 was confirmed by yeast two-hybrid and BiFC assays. The VvGAI1 gene sequence was cloned into the prokaryotic expression vector to form a pET28b-VvGAI1 recombinant vector, and the Escherichia coli BL21 carrying pET28b-VvGAI1 recombinant vector was incubated in culture medium with 1.0 mmol∙L^-1 isopropyl-β-d-thiogalactoside (IPTG) at 16 ℃ to obtain VvGAI1-His fusion protein. The anti-VvGAI1 polyclonal antibody (rabbit-derived) was prepared by antigen immunization and serum purification and used to specifically detect VvGAI1 protein in Chardonnay grapes. Western blot results showed that the VvGAI1 protein in grape protoplasts under low temperature treatment showed an increasing first and then decreasing trend, which indicated the expression of VvGAI1 protein was induced at a low temperature. For 50 μmol·L^-1 MeJA and 50 μmol·L^-1 GA₃ treatments, compared with the control group exogenous MeJA treatment could improve the cold resistance of grape, whereas GA₃ treatment made grapes more sensitive to cold.a【Conclusion】 Grape VvGAI1 protein was a transcription factor and interacts with VvJAZ9. The VvGAI1 responded to low temperature stress, and exogenous MeJA was able to positively regulate the cold stress, while GA₃ negatively regulated the cold response.

Key words: Vitis vinifera, DELLA protein, GAI1, protein interaction, low temperature, expression pattern

LIU DeShuai, FENG Mei, SUN YuTong, WANG Ye, CHI JingNan, YAO WenKong. Analysis of the Interaction Between VvGAI1 and VvJAZ9 Proteins in Grape and Its Expression Pattern Under Low Temperature[J].Scientia Agricultura Sinica, 2023, 56(15): 2977-2994.

Figures/Tables 13

Table 1

List of primers"

引物名称 Primer name	引物序列Primer sequence (5′→3′)	用途 Purpose
VvGAI1-F	ATGAAGAGGGAGTATCATCATCCTCATC	基因克隆 Gene cloning
VvGAI1-R	TCAGTTGGAGGCAGGTGTGGAG	基因克隆 Gene cloning
pBI221-VvGAI1-F-Xba I	GAGAGAACACGGGGGACTCTAGAATGAAGAGGGAGTATCATCATCCTCATC	亚细胞定位Subcellular localization
pBI221-VvGAI1-R-Xho I	TTACCCATGGTACCCCGCTCGAGGTTGGAGGCAGGTGTGGAGG	亚细胞定位Subcellular localization
pGBKT7-VvGAI1-F-EcoRⅠ	TATGGCCATGGAGGCCGAATTCATGAAGAGGGAGTATCATCATCCTCATC	自激活验证 Self-activation verification
pGBKT7-VvGAI1-R-BamHⅠ	GCCGCTGCAGGTCGACGGATCCTCAGTTGGAGGCAGGTGTGGAG	自激活验证 Self-activation verification
pGBKT7-VvGAI1^∆DELLA-F-EcoRⅠ	TATGGCCATGGAGGCCGAATTCATGGGTAAGGCTCTTTATTCCCATATCGAAC	自激活验证 Self-activation verification
pGBKT7-VvGAI1^∆DELLA-R-BamHⅠ	GCCGCTGCAGGTCGACGGATCCTCAGTTGGAGGCAGGTGTGGAG	自激活验证 Self-activation verification
pGBKT7-VvGAI1^∆GRAS-F-EcoRⅠ	TATGGCCATGGAGGCCGAATTCATGAAGAGGGAGTATCATCATCCTCATC	自激活验证 Self-activation verification
pGBKT7-VvGAI1^∆GRAS-R-BamHⅠ	GCCGCTGCAGGTCGACGGATCCTCATGGAATTGCTTTGAGATCGTAATCCAAAGAC	自激活验证 Self-activation verification
pGADT7-VvGAI1-F-EcoRⅠ	GCCATGGAGGCCAGTGAATTCATGAAGAGGGAGTATCATCATCCTCATC	酵母双杂 Yeast two-hybrid
pGADT7-VvGAI1-R-BamHⅠ	CAGCTCGAGCTCGATGGATCCTCAGTTGGAGGCAGGTGTGGAG	酵母双杂 Yeast two-hybrid
pBI221-CE-VvJAZ9-F-Xba I	GAGAACACGGGGGACTCTAGAATGTCGAGCTCCTCGGATATTGC	BiFC验证 BiFC verification
pBI221-CE-VvJAZ9-R-Xho I	GTACATGGTACCCCGCTCGAGCTTTGGAGATTTGGCAGCCAAGC	BiFC验证 BiFC verification
pBI221-NE-VvGAI1-F-Xba I	GAGAACACGGGGGACTCTAGAATGAAGAGAGACCTCCTAGACAGTTGT	BiFC验证 BiFC verification
pBI221-NE-VvGAI1-R-Xho I	GTACATGGTACCCCGCTCGAGTCAGTTGGAGGCAGGTGTGGAG	BiFC验证 BiFC verification
pET28b-VvGAI1-F-Nde I	TGGTGCCGCGCGGCAGCCATATGAAGAGGGAGTATCATCATCCTC	原核表达Prokaryotic expression
pET28b-VvGAI1-R-Xho I	AGTGGTTGGTGGTGGGGTGCTCGAGTCAGTTGGAGGCAGGTGTGGAG	原核表达Prokaryotic expression

Table 1

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Analysis of the Interaction Between VvGAI1 and VvJAZ9 Proteins in Grape and Its Expression Pattern Under Low Temperature

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