VvMYB14调控葡萄花青素合成及互作蛋白筛选

doi:10.3864/j.issn.0578-1752.2026.09.010

摘要/Abstract

摘要：

【目的】花青素是决定葡萄（Vitis vinifera L.）果实色泽、风味和抗氧化活性的核心次生代谢产物，其合成过程受光信号、转录因子和代谢通路关键基因的多级调控，直接影响葡萄果实的商品价值与营养价值。研究MYB转录因子VvMYB14（NCBI ID：XM_002278747.2.）响应光信号调控花青素合成的作用机制，为解析光信号与次生代谢之间的相互作用，以及提高葡萄果实品质提供理论依据。【方法】以葡萄品种‘黑比诺’‘红地球’、苹果品种‘瑞雪’和本氏烟草为试验材料。通过NCBI数据库BLAST工具检索VvMYB14同源序列，利用MEGA 5.0软件构建系统发育树，采用DNAMAN进行氨基酸序列多重比对；构建VvMYB14过表达载体、启动子融合GUS载体等试验载体，借助PlantCARE数据库预测其启动子顺式作用元件，并通过烟草叶片GUS染色与qRT-PCR检测验证启动子活性；结合苹果与葡萄果皮瞬时转化、烟草稳定转化和葡萄愈伤组织遗传转化体系，通过表型观察与分子检测探究VvMYB14的功能；采用酵母双杂交（Y2H）与荧光素酶互补试验（LCA）筛选并验证VvMYB14的互作蛋白。【结果】VvMYB14与欧洲栎QrMYB14关系最为密切，组织特异性分析表明，VvMYB14主要在葡萄叶和果实中表达。其启动子包含光响应元件（Box4、I-box），光响应后能显著增强启动子活性。且GUS表达量在3个时期（G0、G12、G24）均显著高于对照。功能验证表明，光响应后VvMYB14可促进花青素合成：‘瑞雪’苹果和‘红地球’葡萄果皮瞬时表达后，花青素含量分别为空载对照的2.25和2.1倍；过表达VvMYB14的烟草和葡萄愈伤组织中，花青素合成途径关键基因CHS、F3'H、DFR、UFGT的转录水平均显著上调。互作验证证实，VvMYB14可与赤霉素调节蛋白VvGRP4发生特异性相互作用。【结论】VvMYB14是光响应型转录因子，通过识别启动子光响应元件激活自身表达，进而上调花青素合成途径关键基因的转录，最终促进葡萄花青素积累，并筛选到与其互作蛋白VvGRP4。

关键词: 葡萄（Vitis vinifera L.）, MYB, 光信号, 花青素积累, 蛋白质相互作用

Abstract:

【Objective】Anthocyanins are core secondary metabolites that determine the fruit color, flavor and antioxidant activity of grape (Vitis vinifera L.). Their biosynthesis is regulated at multiple levels by light signals, transcription factors and key genes in the metabolic pathway, which directly affects the commercial and nutritional value of grape fruits. To investigate the regulatory mechanism of MYB transcription factor VvMYB14 (NCBI ID: XM_002278747.2) in response to light signals during anthocyanin biosynthesis, so as to provide a theoretical basis for analyzing the interaction between light signals and secondary metabolism, as well as improving grape fruit quality.【Method】Grape cultivars Pinot Noir and Red Globe, apple cultivar Ruixue, and Nicotiana benthamiana were used as experimental materials. Homologous sequences of VvMYB14 were retrieved by BLAST tool from the NCBI database. A phylogenetic tree was constructed using MEGA 5.0 software, and multiple amino acid sequence alignment was performed with DNAMAN. Experimental vectors including VvMYB14 overexpression vector and promoter-GUS fusion vector were constructed. Cis-acting elements in its promoter were predicted using the PlantCARE database, and promoter activity was verified by GUS staining and qRT-PCR assay in tobacco leaves. Combined with transient transformation in apple and grape peels, stable transformation in tobacco, and genetic transformation in grape calli, the function of VvMYB14 was explored through phenotypic observation and molecular detection. Yeast two-hybrid (Y2H) and luciferase complementation assay (LCA) were used to screen and verify the interacting proteins of VvMYB14.【Result】VvMYB14 was most closely related to QrMYB14 of Quercus robur. Tissue-specific analysis showed that VvMYB14 was mainly expressed in grape leaves and fruits. Its promoter contained light-responsive elements (Box4, I-box), and light treatment significantly enhanced the promoter activity. Meanwhile, GUS expression levels were significantly higher than those of the control at three stages (G0, G12, G24). Functional verification indicated that VvMYB14 promoted anthocyanin biosynthesis in response to light: after transient expression in Ruixue apple and Red Globe grape peels, anthocyanin contents were 2.25-fold and 2.1-fold of the empty vector control, respectively. Transcription levels of key anthocyanin pathway genes CHS, F3′H, DFR and UFGT were significantly up-regulated in VvMYB14- overexpressing tobacco and grape calli. Interaction analysis confirmed that VvMYB14 specifically interacted with VvGRP4, a gibberellin-regulated protein.【Conclusion】VvMYB14 is a light-responsive transcription factor. It activates self-expression by recognizing light-responsive elements in the promoter, and then up-regulates the transcription of key genes in the anthocyanin biosynthetic pathway, ultimately promoting anthocyanin accumulation in grapes. In addition, its interacting protein VvGRP4 was identified.

Key words: grape (Vitis vinifera L.), MYB, light signals, anthocyanin accumulation, protein interaction

马宗桓, 南昕彤, 陈丽珍, 杜云博, 唐星, 李文芳, 毛娟, 陈佰鸿. VvMYB14调控葡萄花青素合成及互作蛋白筛选[J]. 中国农业科学, 2026, 59(9): 1975-1986.

MA ZongHuan, NAN XinTong, CHEN LiZhen, DU YunBo, TANG Xing, LI WenFang, MAO Juan, CHEN BaiHong. VvMYB14 Regulates Anthocyanin Biosynthesis in Grape and Screening of Its Interacting Proteins[J]. Scientia Agricultura Sinica, 2026, 59(9): 1975-1986.

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