葡萄苹果酸脱氢酶MDH基因家族鉴定及VvMDH10基因功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0661

摘要/Abstract

摘要：

目的探究葡萄（Vitis vinifera）苹果酸脱氢酶（malate dehydrogenase, MDH）的基因结构与表达，揭示VvMDH基因的生物学功能，为进一步培育优质葡萄品种提供参考。方法利用生物信息学方法对葡萄11个MDH基因进行鉴定，并分析其蛋白理化性质、基因同源性、染色体位置、保守基序（motif）、基因结构和启动子区域等，实时荧光定量PCR（RT-qPCR）验证葡萄MDH基因家族成员在干旱和盐碱胁迫下的表达模式。利用花序浸染法获得异源过表达VvMDH10的拟南芥植株，进行干旱表型分析，探究VvMDH10对植株抗旱性的影响。结果共鉴定到11个VvMDH基因家族成员不均匀分布在9条染色体上。理化性质分析表明，VvMDH多为碱性蛋白，亚细胞定位预测显示VvMDH分布于细胞质基质、叶绿体和线粒体。系统发育分析将VvMDH成员分为5个亚家族，与拟南芥同源蛋白聚类模式高度保守。同一亚族成员具有相似的基因结构及motif分布。启动子顺式作用元件分析结果显示，VvMDH基因富含光响应、激素应答及干旱等胁迫响应元件，表明VvMDH基因可能受到多种植物激素或胁迫的调控。转录组数据分析显示，VvMDH在不同组织间特异性表达。RT-qPCR结果表明，干旱及盐碱处理能够诱导多个VvMDH基因的表达，尤其在干旱胁迫下，VvMDH10的表达量显著上调。异源过表达VvMDH10的拟南芥离体叶片失水率显著低于野生型，干旱处理后，过表达植株生物量和抗氧化相关酶活性显著高于野生型，体内MDA含量显著低于野生型。结论 VvMDH10基因能够正向调控植株的抗旱性，可作为葡萄抗旱分子育种的基因资源。

关键词: ‘马瑟兰’葡萄, 苹果酸脱氢酶（MDH）, VvMDH10, 抗旱, 耐盐碱, 全基因组鉴定, 过表达

Abstract:

Objective To investigate the gene structure and expression of malate dehydrogenase (MDH) in grapevine (Vitis vinifera), and to provide insights into the biological functions of VvMDH genes and to support further breeding of high-quality grape varieties. Method Bioinformatics approaches were employed to identify 11 MDH genes in grapevine, followed by analyses of their protein physicochemical properties, gene homology, chromosomal locations, conserved motifs, gene structures, and promoter regions. Real-time quantitative PCR (RT-qPCR) was used to validate the expression patterns of VvMDH gene family members under drought and saline-alkali stress conditions. Arabidopsis plants heterologously overexpressing VvMDH10 were obtained using the floral dip method for drought phenotype analysis, to investigate the effect of VvMDH10 on plant drought tolerance. Result A total of 11 VvMDH gene family members were identified, unevenly distributed across 9 chromosomes. Physicochemical analysis revealed that most VvMDH proteins are alkaline. Subcellular localization predictions indicated VvMDH is located in the cytosol, chloroplasts, and mitochondria. Phylogenetic analysis classified the VvMDH members into 5 subfamilies, showing high conservation in clustering patterns with homologous proteins from Arabidopsis. Members of the same subfamily exhibited similar gene structures and motif distributions. Promoter cis-acting element analysis demonstrated that VvMDH genes are enriched with light-responsive, hormone-responsive, and stress-responsive elements (e.g., drought stress), suggesting their potential regulation by various phytohormones or stresses. Transcriptome data revealed tissue-specific expression patterns of VvMDH genes. RT-qPCR results indicated that drought and saline-alkali treatments could induce the expression of multiple VvMDH genes, with VvMDH10 expression significantly upregulated especially under drought stress. Detached Arabidopsis thaliana leaves heterologously overexpressing VvMDH10 exhibited a significantly lower water loss rate compared to wild-type plants. Under drought stress, the overexpressing lines showed significantly higher biomass and activities of antioxidant-related enzymes, significantly lower malondialdehyde (MDA) content compared to the wild type. Conclusion The VvMDH10 gene can positively regulate plant drought tolerance and can serve as a genetic resource for molecular breeding of drought-resistant grapevines.

Key words: Marselan grape, malate dehydrogenase (MDH), VvMDH10, drought resistance, salt-alkali tolerance, genome-wide identification, overexpression

高亮, 崔鑫源, 刘名原, 韩蕾. 葡萄苹果酸脱氢酶MDH基因家族鉴定及VvMDH10基因功能分析[J]. 生物技术通报, 2026, 42(10): 142-152.

GAO Liang, CUI Xin-yuan, LIU Ming-yuan, HAN Lei. Identification of Malate Dehydrogenase MDH Gene Family and Functional Characterization of VvMDH10 in Grape[J]. Biotechnology Bulletin, 2026, 42(10): 142-152.

图/表 9

图1 VvMDH基因染色体定位

Fig. 1 Chromosome localization of VvMDH genes in grape

图2 葡萄（Vv）、拟南芥（At）、水稻（Os）、番茄（Sl）和杨树（Pt）MDH蛋白系统进化树

Fig. 2 Phylogenetic tree of MDH proteins in Vitis vinifera (Vv), Arabidopsis thaliana (At), Oryza sativa (Os), Solanum lycopersicum (Sl) and Populus trichocarpa (Pt)

图3 VvMDH基因系统进化树（A）、基因结构（B）和蛋白保守基序（C）

Fig. 3 Phylogenetic tree (A), gene structure (B), and protein conserved motif (C) of VvMDH gene family

图4 VvMDH启动子区顺式作用元件分析

Fig. 4 Analysis of cis-acting elements in the promoter region of VvMDH

图5 VvMDH基因在不同组织中的表达分析

Fig. 5 Expression analysis of VvMDH in different tissues of grape

图6 VvMDH基因在干旱处理下的相对表达量不同小写字母表示差异显著（P<0.05）。下同

Fig. 6 Relative expression of VvMDH genes under drought treatmentsDifferent lower letters indicate significant differences (P<0.05). The same below

图7 VvMDH基因在盐碱处理下的相对表达量

Fig. 7 Relative expression of VvMDH genes under salt-alkali treatments

图8 VvMDH10在转基因拟南芥中的相对表达量

Fig. 8 Relative expression of VvMDH10 in transgenic A. thaliana

图9 干旱胁迫下VvMDH10异源过表达转基因拟南芥的表型和生理指标比较将拟南芥野生型及过表达株系置于含有泥炭土的营养钵中培养4周，先将整个营养钵充分灌水，然后停止浇水，观测植物干旱处理前后的表型，测定丙二醛含量和多种酶活性。后将拟南芥野生型及过表达株系置于含有泥炭土的营养钵中培养4周，分别剪取其整个莲座叶，置于培养皿中，放入光照培养箱，每隔一段时间称取其鲜重。A：植株表型；B：地上部鲜重；C：SOD活性；D：丙二醛含量；E：POD活性；F：CAT活性；G：叶片失水表型；H：叶片失水率统计；图B‒F、H中数据为平均值±标准差

Fig. 9 Comparisons of phenotype and physiological indices in VvMDH10-overexpressing Arabidopsis under drought stressArabidopsis thaliana wild-type and overexpression lines were grown in pots containing peat soil for 4 weeks. The entire pots were fully watered first, then watering was stopped. The phenotypes of the plants before and after drought treatment were observed, and MDA content and activities of various enzymes were determined. Then the Arabidopsis thaliana wild-type and overexpression lines were grown in pots containing peat soil for 4 weeks, and their entire rosette leaves were cut separately, placed in petri dishes, and put into an illumination incubator. The fresh weight was measured at regular intervals. A: Plant phenotype. B: Shoot fresh weight. C: SOD activity. D: MDA content. E: POD activity. F: CAT activity. G: Leaf water loss phenotype. H: Statistical analysis of leaf water loss rate. Data in Figures B-F and H are presented as mean ± standard deviation

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