酿酒葡萄VvOMTs基因家族鉴定及启动子功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0459

摘要/Abstract

摘要：

目的鉴定葡萄（Vitis vinifera L.）VvOMTs基因家族启动子对激素和干旱信号的响应，阐明VvOMTs基因家族的启动子功能，为干旱条件下VvOMTs调控3-烷基-2-甲氧基吡嗪香气物质合成提供理论依据。方法采用生物信息学方法分析其蛋白理化性质、染色体定位、基因结构和启动子顺式作用元件；通过农杆菌介导的烟草瞬时转化体系验证VvOMT2和VvOMT3基因启动子对脱落酸（abscisic acid, ABA）、茉莉酸酸甲酯（methyl jasmonate, MeJA）和甘露醇的响应。结果葡萄VvOMTs基因家族编码序列CDS区介于1 062‒1 110 bp，蛋白氨基酸数介于353‒369 aa；4个VvOMTs在葡萄第3和第12号染色体上均匀分布；亚细胞定位预测结果表明，VvOMTs蛋白存在于细胞质；启动子顺式作用元件分析表明，VvOMTs基因可能受到光照、多种植物激素和干旱胁迫的诱导；瞬时转化本氏烟草GUS染色、GUS基因定量和酶活活性测定结果表明，在ABA、MeJA和甘露醇的诱导下，转化VvOMT2和VvOMT3启动子的烟草叶片均呈现蓝色反应；VvOMT2和VvOMT3的GUS基因表达水平呈现先升高后下降趋势，且显著上调16.80和77.73倍、14.09和34.15倍、30.77和36.40倍；VvOMT2和VvOMT3的GUS酶活性在ABA和MeJA处理下呈先升高后下降，且显著上调16.62和21.69倍、4.50和3.00倍，甘露醇处理下出现“双峰”变化趋势，显著上调15.13和18.64倍。结论从葡萄基因组中共鉴定出4个调控甲氧基吡嗪合成的甲氧基转移酶基因（VvOMT1-4），VvOMT2和VvOMT3基因启动子参与干旱、ABA和MeJA响应，并受其调控。

关键词: 酿酒葡萄, VvOMTs基因家族, 生物信息学分析, 启动子功能, 激素处理, 干旱胁迫, 甲氧基吡嗪

Abstract:

Objective To characterize the response of grape (Vitis vinifera L.) VvOMTs gene family promoters to hormone and drought signals, to elucidate the promoters functions of the VvOMTs gene family, and to provide a theoretical basis for the regulation of the synthesis of 3-alkyl-2-methoxypyrazine aroma substances by VvOMTs under drought conditions. Method Bioinformatics methods were used to analyze the physicochemical properties of the protein, chromosomal localization, gene structure, and promoters cis-acting elements; and the promoters of the VvOMT2/3 gene was verified by an Agrobacterium-mediated transient transformation system in tobacco in response to abscisic acid (ABA), methyl jasmonate (MeJA), and mannitol. Result The CDS region of the coding sequence of the grape VvOMTs gene family ranged from 1 062‒1 110 bp, and the number of protein amino acids ranged in353-369 aa; the four VvOMTs were evenly distributed on the two grape chromosome 3 and 12; subcellular localization predictions indicated that the VvOMTs proteins existed in the cytoplasm; the promoters cis-acting element analysis indicated that the VvOMTs genes might be subjected to induction by light, multiple plant hormones and drought stress. The results of GUS staining, gene quantification and enzyme activity assay of transiently transformed tobacco showed that under the induction of ABA, MeJA and mannitol, the tobacco leaves transformed with VvOMT2/3 promoters showed a blue color reaction, and the expression of the GUS gene of VvOMT2/3 significantly increased and showed a tendency of increasing and then decreasing, and the GUS enzyme activity of VvOMT2/3 significantly increased. The GUS enzyme activity of VvOMT2/3 significantly increased and showed a trend of increasing and then decreasing under hormone treatment, and a bimodal trend under drought treatment. Conclusion A total of four methoxytransferase genes (VvOMT1-4) regulating methoxypyrazine synthesis are identified from the grape genome, and the VvOMT2 and VvOMT3 gene promoters are involved in and regulated by drought, ABA and MeJA responses.

Key words: wine grape, VvOMTs gene family, bioinformatics analysis, promoters function, hormone treatment, drought stress, methoxypyrazine

薛晓斌, 宁琳, 周鱼, 刘虹君, 高照祖, 王振平, 李栋梅. 酿酒葡萄VvOMTs基因家族鉴定及启动子功能分析[J]. 生物技术通报, 2025, 41(12): 168-176.

XUE Xiao-bin, NING Lin, ZHOU Yu, LIU Hong-jun, GAO Zhao-zu, WANG Zhen-ping, LI Dong-mei. Identification of VvOMTs Gene Families and Functional Analysis of Promoters in Wine Grapes[J]. Biotechnology Bulletin, 2025, 41(12): 168-176.

图/表 7

表1 葡萄VvOMTs家族基因扩增和RT-qPCR引物序列

Table 1 Amplification and RT-qPCR primer sequences for grape (Vitis vinifera L.) VvOMTs family genes

基因名称 Gene name	上游引物序列 Forward primer （5′-3′）	下游引物序列 Reverse primer （5′-3′）
pC2301-ProVvOMT2	gctatgaccatgattacgaattcGACACAAATTAAGATGCCGG	atggggtcgactctagaggatcccatTTTCTGCTTCTCTCTTATGGAG
pC2301-ProVvOMT3	gctatgaccatgattacgaattcGGCCCTATACAAATTGCCCAG	atggggtcgactctagaggatcccatCAATGGCACACTTCACTACAG
RT-GUS	GCTCTACACCACGCCGAACA	CCACCACCTGCCAGTCAACA
RT-Ntactin	TTGCCTGATGGACAAGTTATTACC	TAGGAGCCAAAGCCGTGATT

表2 葡萄VvOMTs家族成员蛋白理化性质分析

Table 2 Analysis of physical and chemical properties of grape VvOMTs family member proteins

基因编号 Gene No.	基因名称 Gene name	编码序列 CDS sequence （bp）	氨基酸数量 Number of amino acids （aa）	分子质量 Molecular weight （kD）	理论等电点 pI	亚细胞定位Subcellular localization
KC533532.1	VvOMT1	1 110	369	40 835.11	5.21	细胞质
KC533540.1	VvOMT2	1 104	367	40 312.59	5.53	细胞质
KC243502.1	VvOMT3	1 062	353	38 595.60	5.70	细胞质
KC517476.1	VvOMT4	1 080	359	39 263.11	5.09	细胞质

图1 VvOMTs家族成员染色体定位（A）和基因结构分析（B-E）

Fig. 1 Chromosome localization (A) and gene structure analysis (B-E) of VvOMTs family members

图2 葡萄VvOMTs家族成员上游启动子序列顺式作用元件分析

Fig. 2 Analysis of cis-acting elements in the upstream promoters' region of grape VvOMTs family members

图3 ‘马瑟兰’葡萄VvOMTs基因启动子对ABA的响应A：空载体、VvOMT2/3基因启动子在ABA处理后烟草叶片的GUS染色，bar=1 cm；B：GUS基因相对表达水平；C：GUS蛋白酶活活性。** P≤0.01，*** P≤0.001，下同

Fig. 3 ‘Marselan’ grape VvOMTs gene promoters' response to ABAA: GUS staining of tobacco leaves after ABA treatment with empty vector, VvOMT2/3 gene promoters, bar=1 cm. B: Relative expression of GUS gene. C: GUS protease activity. ** P≤0.01, *** P≤0.001. The same below

图4 ‘马瑟兰’葡萄VvOMTs基因启动子对甘露醇的响应A：空载体、VvOMT2/3基因启动子在甘露醇处理后烟草叶片的GUS染色，bar=1 cm；B：GUS基因相对表达水平；C：GUS蛋白酶活活性

Fig. 4 ‘Marselan’ grape VvOMTs gene promoters' responses to mannitolA: GUS staining of tobacco leaves after mannitol treatment with empty vector, VvOMT2/3 gene promoters, bar=1 cm. B: Relative expression level of GUS gene. C: GUS protease activity

图5 ‘马瑟兰’葡萄VvOMTs基因启动子对MeJA的响应A：空载体、VvOMT2/3基因启动子在MeJA处理后烟草叶片的GUS染色，bar=1 cm；B：GUS基因相对表达水平；C：GUS蛋白酶活活性

Fig. 5 ‘Marselan’ grape VvOMTs gene promoters' responses to MeJAA: GUS staining of tobacco leaves after MeJA treatment with empty vector, VvOMT2/3 gene promoters, bar=1 cm. B: Relative expression level of GUS gene. C: GUS protease activity

参考文献 26

[1]	Seifert RM, Buttery RG, Guadagni DG, et al. Synthesis of some 2-methoxy-3-alkylpyrazines with strong bell pepper-like odors [J]. J Agric Food Chem, 1970, 18(2): 246-249.
[2]	张艳霞, 吕丹桂, 耿康奇, 等. 水分胁迫对赤霞珠葡萄果实品质和甲氧基吡嗪含量的影响 [J]. 果树学报, 2022, 39(6): 1017-1028.
	Zhang YX, Lü DG, Geng KQ, et al. Effects of water stress on grape quality and content of methoxypyrazines in Cabernet Sauvignon [J]. J Fruit Sci, 2022, 39(6): 1017-1028.
[3]	Hashizume K, Tozawa K, Endo M, et al. S-Adenosyl-L-methionine-dependent O-methylation of 2-hydroxy-3-alkylpyrazine in wine grapes: a putative final step of methoxypyrazine biosynthesis [J]. Biosci Biotechnol Biochem, 2001, 65(4): 795-801.
[4]	Sidhu D, Lund J, Kotseridis Y, et al. Methoxypyrazine analysis and influence of viticultural and enological procedures on their levels in grapes, musts, and wines [J]. Crit Rev Food Sci Nutr, 2015, 55(4): 485-502.
[5]	Sala C, Mestres M, Martı́ MP, et al. Headspace solid-phase microextraction analysis of 3-alkyl-2-methoxypyrazines in wines [J]. J Chromatogr A, 2002, 953(1/2): 1-6.
[6]	Roujou de Boubée D, Van Leeuwen C, Dubourdieu D. Organoleptic impact of 2-methoxy-3-isobutylpyrazine on red Bordeaux and Loire wines. Effect of environmental conditions on concentrations in grapes during ripening [J]. J Agric Food Chem, 2000, 48(10): 4830-4834.
[7]	Zhang YX, Li XY, Guo XF, et al. Comparison of methoxypyrazine content and expression pattern of O-methyltransferase genes in grape berries and wines from six cultivars (Vitis vinifera L.) in the eastern foothill of the Helan Mountain [J]. Plants, 2022, 11(12): 1613.
[8]	来泽森. 拟南芥转录因子bHLH010和bHLH089调控花药和花粉发育的机制研究 [D]. 海口: 海南大学, 2022.
	Lai ZS. Mechanism of transcription factor bHLH010 and bHLH089 regulating anther and pollen development in Arabidopsis thaliana [D]. Haikou: Hainan University, 2022.
[9]	戴梦怡, 郑钢, 程少禹, 等. 紫玉兰MlOMT1基因的克隆和表达分析 [J]. 分子植物育种, 2022, 20(16): 5267-5273.
	Dai MY, Zheng G, Cheng SY, et al. Cloning and expression analysis of MlOMT1 gene in Magnolia liliflora [J]. Mol Plant Breed, 2022, 20(16): 5267-5273.
[10]	Peng ZX, Song LZ, Chen MH, et al. Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus [J]. Proc Natl Acad Sci USA, 2024, 121(14): e2321615121.
[11]	张帆. 连翘苷生物合成途径OMT候选基因功能研究 [D]. 泰安: 山东农业大学, 2024.
	Zhang F. Functional study of OMT candidate genes in the biosynthetic pathway of forsythoside [D]. Tai’an: Shandong Agricultural University, 2024.
[12]	Chang JJ, Guo YL, Yan JY, et al. The role of watermelon caffeic acid O-methyltransferase (ClCOMT1) in melatonin biosynthesis and abiotic stress tolerance [J]. Hortic Res, 2021, 8(1): 210.
[13]	闫丽. 外源葡萄糖对牡丹品种‘洛阳红’与‘太阳’切花花青素苷合成的影响研究 [D]. 北京: 北京林业大学, 2021.
	Yan L. Effects of exogenous glucose on anthocyanin glycosides synthesis of peony cultivars ‘Luoyanghong’ and ‘Taiyang’ cut flowers [D]. Beijing: Beijing Forestry University, 2021.
[14]	Li DM, Zhang YX, Geng KQ, et al. Impact of vine water status on 3-alkyl-2-methoxypyrazine content and function verification of VvOMT2/VvOMT3 genes associated with 3-alkyl-2-methoxypyrazine accumulation in marselan grape berries (Vitis vinifera L.) [J]. J Agric Food Chem, 2023, 71(49): 19288-19301.
[15]	Guo SH, Yang BH, Wang XW, et al. ABA signaling plays a key role in regulated deficit irrigation-driven anthocyanins accumulation in ‘Cabernet Sauvignon’ grape berries [J]. Environ Exp Bot, 2021, 181: 104290.
[16]	Jefferson RA. Assaying chimeric genes in plants: the GUS gene fusion system [J]. Plant Mol Biol Report, 1987, 5(4): 387-405.
[17]	徐伟荣. 中国华东葡萄抗白粉病芪合成酶基因及启动子克隆与功能分析 [D]. 杨凌: 西北农林科技大学, 2010.
	Xu WR. Cloning and functional analysis of stilbene synthase and its promoter from powdery mildew-resistant Chinese Vitis pseudoreticulata [D]. Yangling: Northwest A & F University, 2010.
[18]	Inoue K, Sewalt VJ, Murray GB, et al. Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification [J]. Plant Physiol, 1998, 117(3): 761-770.
[19]	Kan YB, Xie SY, Sun YW, et al. Substrate and functional characterization of the lysine acetyltransferase MsKat and deacetylase MsCobB in Mycobacterium smegmatis [J]. J Proteom, 2024, 300: 105177.
[20]	Li SL, Cai YT, Ma HM, et al. A comprehensive analysis of the O-methyltransferase gene family in the chili pepper (Capsicum annuum) identifies COMT36 involved in capsaicinoids biosynthesis [J]. Int J Biol Macromol, 2025, 312: 144247.
[21]	Ke LP, Yu DL, Zheng HL, et al. Function deficiency of GhOMT1 causes anthocyanidins over-accumulation and diversifies fibre colours in cotton (Gossypium hirsutum) [J]. Plant Biotechnol J, 2022, 20(8): 1546-1560.
[22]	Torres-Díaz LL, Pérez-Álvarez EP, Parra-Torrejón B, et al. Effects of foliar application of methyl jasmonate and/or urea, conventional or via nanoparticles, on grape volatile composition [J]. J Sci Food Agric, 2024, 104(13): 8248-8262.
[23]	陈祖民. 水分胁迫对‘美乐’葡萄不同组织内源激素和多胺的影响 [D]. 银川: 宁夏大学, 2021.
	Chen ZM. Effects of water stress on endogenous hormones and polyamines in different tissues of ‘Merlot’ grape [D]. Yinchuan: Ningxia University, 2021.
[24]	Ma JB, Wei LR, Huang KY, et al. Biosynthesis of sakuranetin regulated by OsMPK6-OsWRKY67-OsNOMT cascade enhances resistance to false smut disease [J]. New Phytol, 2025, 245(3): 1216-1231.
[25]	王晓宇. 小麦非生物胁迫相关LEA蛋白WZY1-2与WRAB18的功能研究 [D]. 杨凌: 西北农林科技大学, 2017.
	Wang XY. Functional research of abiotic stress-related LEA proteins WZY1-2 and WRAB18 in wheat (Triticum aestivum) [D]. Yangling: Northwest A & F University, 2017.
[26]	李攀. 拟南芥和玉米糖基转移酶基因参与非生物胁迫耐性的功能研究 [D]. 济南: 山东大学, 2017.
	Li P. Functional analysis of glycosyltransferase genes of Arabidopsis thaliana and Zea mays in tolerance to abiotic stresses [D]. Jinan: Shandong University, 2017.