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

摘要/Abstract

摘要：

【目的】 DELLA蛋白属于植物特有的GRAS蛋白家族，是赤霉素信号转导途径中的重要调控因子，在植物生长发育和抵御逆境胁迫中发挥着重要作用。克隆欧洲葡萄VvGAI1，并对其进行亚细胞定位、蛋白互作和表达分析，为进一步研究DELLA蛋白在葡萄抗寒反应中的调控功能奠定基础。【方法】以酿酒葡萄品种‘霞多丽'叶片为试材，采用同源克隆的方法获得VvGAI1序列。利用生物信息学方法分析该基因的序列特征，使用DNAMAN及MEGA7.0对VvGAI1蛋白序列及拟南芥序列进行多序列比对并构建系统进化树。通过亚细胞定位确定VvGAI1蛋白在细胞中的表达位置；利用酵母试验验证VvGAI1蛋白的转录激活活性；利用酵母双杂交和双分子荧光互补试验验证VvGAI1蛋白与VvJAZ9蛋白的互作关系；利用VvGAI1原核表达蛋白制备anti-VvGAI1兔源多克隆抗体；利用Western Blot技术检测VvGAI1蛋白在低温下的表达情况；通过相对电导率分析外源喷施茉莉酸和赤霉素对葡萄抗寒性的影响。【结果】从‘霞多丽'叶片中克隆得到VvGAI1，其ORF为1 773 bp，编码590个氨基酸，位于第1条染色体，含有1个外显子，无内含子。VvGAI1蛋白相对分子质量为64.87 kDa，理论等电点pI为5.31，属于酸性不稳定亲水蛋白。VvGAI1具有高度保守的DELLA和GRAS结构域，属于植物GRAS家族的DELLA蛋白。蛋白聚类分析显示VvGAI1与拟南芥AtGAI和烟草NtGAI1亲缘关系较近。亚细胞定位与转录自激活结果显示VvGAI1是一个定位于细胞核中且具有转录激活活性的转录因子。酵母双杂交和双分子荧光互补试验也证实VvGAI1与VvJAZ9具有互作关系。将VvGAI1克隆至原核表达载体构建pET28b-VvGAI1重组表达载体，转化至大肠杆菌BL21中经16 ℃、1.0 mmol∙L^-1异丙基-β-d-硫代半乳糖苷（IPTG）诱导表达获得VvGAI1-His融合蛋白，再经抗原免疫，血清纯化后制备得到anti-VvGAI1兔源多克隆抗体。制备的anti-VvGAI1抗体可以特异性检测‘霞多丽'葡萄中的VvGAI1蛋白。Western Blot结果表明，低温处理下葡萄原生质体中VvGAI1蛋白呈先上升后下降的表达趋势，VvGAI1蛋白响应低温诱导表达。与对照相比，50 μmol·L^-1茉莉酸甲酯（MeJA）处理可以提高葡萄的抗寒性，50 μmol·L^-1赤霉素（GA₃）处理使葡萄对寒冷变得敏感。【结论】葡萄VvGAI1是一个与VvJAZ9相互作用的转录因子，VvGAI1对低温胁迫有响应，外源茉莉酸能够正调控冷胁迫反应，而赤霉素负调控冷胁迫反应。

关键词: 葡萄, DELLA蛋白, GAI1, 蛋白互作, 低温, 表达模式

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

刘德帅, 冯美, 孙雨桐, 王烨, 迟敬楠, 姚文孔. 葡萄VvGAI1与VvJAZ9蛋白互作及低温下的表达模式分析[J]. 中国农业科学, 2023, 56(15): 2977-2994.

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.

图/表 13

表1

引物列表"

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

表1

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