不同葡萄种质果皮中香气物质含量及基因表达的差异分析

doi:10.3864/j.issn.0578-1752.2024.13.011

摘要/Abstract

摘要：

【目的】探究不同葡萄种质成熟期果皮中挥发性香气物质种类、含量及相关基因表达量的差异，为葡萄挥发性香气物质代谢调控机制的解析及香气资源的充分利用提供参考依据。【方法】本研究采用气相色谱-质谱联用技术（GC-MS），对13个葡萄种质果皮中挥发性香气物质的种类和含量进行鉴定；通过Illumina高通量测序平台，对其全基因组转录水平进行分析。【结果】 13个葡萄种质中共检测出154种挥发性香气物质，主要包括醛类、酯类、萜类等化合物。果皮中挥发性香气物质含量最高的种质是‘巨峰’，最低的种质是‘赤霞珠’。‘巨峰’和‘小味儿多’果皮中挥发性香气物质含量最多的是酯类化合物，而其他种质果皮中含量最多的是醛类化合物。正己醛、辛酸乙酯、芳樟醇等12种特征香气物质对葡萄果皮香气贡献较大。主成分分析结果显示，‘巨峰’‘无核白葡萄’‘红地球’与其他种质果皮中挥发性香气物质差异较大。转录组分析结果显示，不同种质间的差异表达基因数量存在较大差异。差异表达基因在亚油酸代谢、α-亚麻酸代谢、苯丙氨酸代谢、酪氨酸代谢和单萜生物合成等途径上显著富集。脂氧合酶（LOX）、醇酰基转移酶（AMAT）、脱氧-d-木酮糖-5-磷酸还原异构酶（DXS）、乙醇脱氢酶（ADH）等基因的表达与香气化合物的含量高度相关。转录因子WRKY7、WRKY28、ARF4和ARF22是葡萄香气合成的潜在调控因子。【结论】不同葡萄种质果皮中香气物质含量及相关基因表达量差异较大，多组学联合分析结果可为进一步研究葡萄挥发性香气物质合成机制提供参考。

关键词: 葡萄, 果皮, 香气, 挥发性香气物质, 代谢组, 转录组

Abstract:

【Objective】 This study aimed to explore the differences in the types, content, and related gene expression levels of volatile aroma compounds in the mature grape skins of different grape germplasms, so as to provide a reference basis for the analysis of the metabolic regulation mechanism of grape volatile aroma substances and the full utilization of aroma resources. 【Method】 In this study, GC-MS was used to identify the types and content of volatile aroma substances in the pericarp of 13 grape germplasms. The transcriptional level of the entire genome was also analyzed by using Illumina high-throughput sequencing platform.【Result】 A total of 154 volatile aroma substances were detected in the 13 grape germplasms, mainly including aldehydes, esters, terpenoids and other compounds. The germplasm with the highest content of volatile aroma substances in the skin was Kyoho, while the lowest was Cabernet Sauvignon. Esters were the most abundant volatile aroma substances in the skins of Kyoho and Petit Verdot, while aldehydes were the most abundant volatile aroma substances in the other germplasms skins. Twelve characteristic aroma substances such as hexanal, ethyl caprylate and linalool contributed more to the aroma of grape skins. The results of principal component analysis showed that there were large differences in volatile aroma substances in the skins of Kyoho, Thompson Seedless, Red Globe and other germplasm. The results of transcriptome analysis showed that the number of differentially expressed genes (DEGs) varied considerably among different germplasms. DEGs were significantly enriched in the pathways of linoleic acid metabolism, α-linolenic acid metabolism, phenylalanine metabolism, tyrosine metabolism, and monoterpene biosynthesis. The expressions of lipoxygenase (LOX), alcohol acyltransferase (AMAT), deoxyd-xylose-5-phosphate reductase (DXS), alcohol dehydrogenase (ADH) and other genes were highly correlated with the content of aroma compounds. Further analysis revealed that the transcription factors WRKY7, WRKY28, ARF4, and ARF22 were potential regulators of aroma synthesis in grapes.【Conclusion】 The content of aroma substances and the expression of related genes in the pericarp of different grape germplasm varied greatly, and the results of the joint multi-omics analysis provided a reference for further research on the synthesis mechanism of volatile aroma substances in grapes.

Key words: grape (Vitis vinifera), skin, aroma, volatile aroma substance, metabolome, transcriptome

徐梦雨, 王佳洋, 王江波, 唐雯, 陈一恒, 上官凌飞, 房经贵, 卢素文. 不同葡萄种质果皮中香气物质含量及基因表达的差异分析[J]. 中国农业科学, 2024, 57(13): 2635-2650.

XU MengYu, WANG JiaYang, WANG JiangBo, TANG Wen, CHEN YiHeng, SHANGGUAN LingFei, FANG JingGui, LU SuWen. Differential Analysis of Aroma Substance Content and Gene Expression in the Berry Skins of Different Grape Germplasms[J]. Scientia Agricultura Sinica, 2024, 57(13): 2635-2650.

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参考文献 39

[1]	MATEO J J, JIMÉNEZ M. Monoterpenes in grape juice and wines. Journal of Chromatography A, 2000, 881(1/2): 557-567.
[2]	刘翠霞. 葡萄果实单萜化合物含量的QTL定位及其合成调控的候选基因筛选[D]. 武汉: 中国科学院武汉植物园, 2017.
	LIU C X. QTL mapping of monoterpene content in grape berrry and screening of candidate genes related to regulation[D]. Wuhan: Wuhan Botanical Garden, Chinese Academy of Sciences, 2017. (Chinese)
[3]	BARBERA D, AVELLONE G, FILIZZOLA F, MONTE L G, CATANZARO P, AGOZZINO P. Determination of terpene alcohols in Sicilian Muscat wines by HS-SPME-GC-MS. Natural Product Research, 2013, 27(6): 541-547. doi: 10.1080/14786419.2012.676553 pmid: 22502739
[4]	YANG C X, WANG Y J, LIANG Z C, FAN P G, WU B H, YANG L, WANG Y N, LI S H. Volatiles of grape berries evaluated at the germplasm level by headspace-SPME with GC-MS. Food Chemistry, 2009, 114(3): 1106-1114.
[5]	DUDAREVA N, PICHERSKY E, GERSHENZON J. Biochemistry of plant volatiles. Plant Physiology, 2004, 135(4): 1893-1902. doi: 10.1104/pp.104.049981 pmid: 15326281
[6]	DEFILIPPI B G, MANRÍQUEZ D, LUENGWILAI K, GONZÁLEZ- AGÜERO M. Aroma volatiles: Biosynthesis and mechanisms of modulation during fruit ripening. Advances in Botanical Research, 2009, 50: 1-37.
[7]	李海燕. ‘阳光玫瑰’葡萄香气物质积累规律及其调控研究[D]. 杭州: 浙江大学, 2017.
	LI H Y. The research of the volatile aroma accumulation and regulation of ‘Shine Muscat’ grape[D]. Hangzhou: Zhejiang University, 2017. (in Chinese)
[8]	姜文广, 李记明, 徐岩, 范文来, 于英. 4种酿酒红葡萄果实的挥发性香气成分分析. 食品科学, 2011, 32(6): 225-229. doi: 10.7506/spkx1002-6630-201106052
	JIANG W G, LI J M, XU Y, FAN W L, YU Y. Analysis of aroma components in four red grape varieties. Food Science, 2011, 32(6): 225-229. (in Chinese)
[9]	满坤. 不同光质对‘阳光玫瑰’葡萄香气组分及风味的影响[D]. 杭州: 浙江大学, 2020.
	MAN K. Effect of different light quality on aroma components and flavor of ‘Shine Muscat’ grape[D]. Hangzhou: Zhejiang University, 2020. (in Chinese)
[10]	杨晓帆, 高媛, 韩梅梅, 彭振雪, 潘秋红. 云南高原区酿酒葡萄果实香气物质的积累规律. 中国农业科学, 2014, 47(12): 2405-2416. doi: 10.3864/j.issn.0578-1752.2014.12.013.
	YANG X F, GAO Y, HAN M M, PENG Z X, PAN Q H. Accumulation characteristics of volatile compounds in wine grape berries grown in high altitude regions of Yunnan. Scientia Agricultura Sinica, 2014, 47(12): 2405-2416. doi: 10.3864/j.issn.0578-1752.2014.12.013. (in Chinese)
[11]	殷梦婷, 代红军, 贺琰, 汪月宁, 郭学良, 刘妍, 王振平. 水分胁迫对马瑟兰葡萄果实挥发性物质合成的影响. 果树学报, 2023, 40(8): 1592-1605.
	YIN M T, DAI H J, HE Y, WANG Y N, GUO X L, LIU Y, WANG Z P. Effects of water stress on the synthesis of volatile compounds in Marselan grape berries. Journal of Fruit Science, 2023, 40(8): 1592-1605. (in Chinese)
[12]	张克坤, 陈可钦, 李婉平, 乔浩蓉, 张俊霞, 刘凤之, 房玉林, 王海波. 灌水量对限根栽培‘阳光玫瑰’葡萄果实发育与香气物质积累的影响. 中国农业科学, 2023, 56(1): 129-143. doi: 10.3864/j.issn. 0578-1752.2023.01.010.
	ZHANG K K, CHEN K Q, LI W P, QIAO H R, ZHANG J X, LIU F Z, FANG Y L, WANG H B. Effects of irrigation amount on berry development and aroma components accumulation of Shine Muscat grape in root-restricted cultivation. Scientia Agricultura Sinica, 2023, 56(1): 129-143,doi: 10.3864/j.issn.0578-1752.2023.01.010. (Chinese)
[13]	孙磊, 王晓玥, 王慧玲, 闫爱玲, 张国军, 任建成, 徐海英. 不同砧木对鲜食葡萄生长和香气品质的影响. 中国农业科学, 2021, 54(20): 4405-4429. doi: 10.3864/j.issn.0578-1752.2023.01.010.
	SUN L, WANG X Y, WANG H L, YAN A L, ZHANG G J, REN J C, XU H Y. The influence of rootstocks on the growth and aromatic quality of two table grape varieties. Scientia Agricultura Sinica, 2021, 54(20): 4405-4429. doi: 10.3864/j.issn.0578-1752.2023.01.010. (in Chinese)
[14]	张乐, 张亚红, 乔振羽, 王亚楠, 陈璐, 周娟, 黄嘉俊. 不同昼夜温差对赤霞珠葡萄果实香气的影响及转录组分析. 核农学报, 2023, 37(4): 865-878. doi: 10.11869/j.issn.1000-8551.2023.04.0865
	ZHANG L, ZHANG Y H, QIAO Z Y, WANG Y N, CHEN L, ZHOU J, HUANG J J. Effect of diurnal amplitude on fruit aroma of Cabernet sauvignon and transcriptome analysis. Journal of Nuclear Agricultural Sciences, 2023, 37(4): 865-878. (in Chinese) doi: 10.11869/j.issn.1000-8551.2023.04.0865
[15]	张文文. 鲜食葡萄香气特征与感官品评的研究[D]. 上海: 上海交通大学, 2018.
	ZHANG W W. Study on the characteristics of aroma and sensory evaluation of table grapes[D]. Shanghai: Shanghai Jiao Tong University, 2018. (in Chinese)
[16]	DALLA COSTA L, EMANUELLI F, TRENTI M, MORENO-SANZ P, LORENZI S, COLLER E, MOSER S, SLAGHENAUFI D, CESTARO A, LARCHER R, GRIBAUDO I, COSTANTINI L, MALNOY M, GRANDO M S. Induction of terpene biosynthesis in berries of microvine transformed with VvDXS1 alleles. Frontiers in Plant Science, 2017, 8: 2244.
[17]	ZHU B Q, XU X Q, WU Y W, DUAN C Q, PAN Q H. Isolation and characterization of two hydroperoxide lyase genes from grape berries: HPL isogenes in Vitis vinifera grapes. Molecular Biology Reports, 2012, 39(7): 7443-7455.
[18]	MAGGU M, WINZ R, KILMARTIN P A, TROUGHT M C T, NICOLAU L. Effect of skin contact and pressure on the composition of Sauvignon Blanc must. Journal of Agricultural and Food Chemistry, 2007, 55(25): 10281-10288. pmid: 18020411
[19]	LI Y Z, HE L L, SONG Y H, ZHANG P, CHEN D D, GUAN L P, LIU S J. Comprehensive study of volatile compounds and transcriptome data providing genes for grape aroma. BMC Plant Biology, 2023, 23(1): 171. doi: 10.1186/s12870-023-04191-1 pmid: 37003985
[20]	孙宝国, 刘玉平, 郑福平. 食用调香术. 2版. 北京: 化学工业出版社, 2010.
	SUN B G, LIU Y P, ZHENG F P. Edible Flavoring. 2nd ed. Beijing: Chemical Industry Press, 2010. (in Chinese)
[21]	PALMA-HARRIS C, MCFEETERS R F, FLEMING H P. Fresh cucumber flavor in refrigerated pickles: Comparison of sensory and instrumental analysis. Journal of Agricultural and Food Chemistry, 2002, 50(17): 4875-4877.
[22]	CABRITA M J, FREITAS A M C, LAUREANO O, DI STEFANO R,. Glycosidic aroma compounds of some Portuguese grape cultivars. Journal of the Science of Food and Agriculture, 2006, 86(6): 922-931.
[23]	张明霞, 吴玉文, 段长青. 葡萄与葡萄酒香气物质研究进展. 中国农业科学, 2008, 41(7): 2098-2104. doi: 10.3864/j.issn.0578-1752. 2008.07.030.
	ZHANG M X, WU Y W, DUAN C Q. Progress in study of aromatic compounds in grape and wine. Scientia Agricultura Sinica, 2008, 41(7): 2098-2104. doi: 10.3864/j.issn.0578-1752.2008.07.030. (in Chinese)
[24]	CANOSA P, OLIVEIRA J M, MASA A, VILANOVA M. Study of the volatile and glycosidically bound compounds of minority Vitis vinifera red cultivars from NW Spain. Journal of the Institute of Brewing, 2011, 117(3): 462-471.
[25]	YANG C X, WANG Y J, WU B H, FANG J B, LI S H. Volatile compounds evolution of three table grapes with different flavour during and after maturation. Food Chemistry, 2011, 128(4): 823-830.
[26]	孙婷. 草莓香型葡萄果实香气物质分析与QTL定位[D]. 沈阳: 沈阳农业大学, 2019.
	SUN T. QTL mapping and study for labrusca flavor compounds in grape[D]. Shenyang: Shenyang Agricultural University, 2019. (in Chinese)
[27]	张文文, 吴玉森, 陈毓谨, 郑奇志, 马超, 许文平, 张才喜, 王世平. 3种巨峰系葡萄的香气特征. 上海交通大学学报(农业科学版), 2018, 36(5): 51-59, 66.
	ZHANG W W, WU Y S, CHEN Y J, ZHENG Q Z, MA C, XU W P, ZHANG C X, WANG S P. Aroma characteristics of three Kyoho grapevine series. Journal of Shanghai Jiao Tong University (Agricultural Science), 2018, 36(5): 51-59, 66. (in Chinese)
[28]	韩洁, 郑艳, 陈存坤, 董成虎, 王文生. 不同贮藏条件对无核白葡萄香气组分变化及品质的影响. 食品与发酵工业, 2015, 41(9): 181-187.
	HAN J, ZHENG Y, CHEN C K, DONG C H, WANG W S. Effects of different storage conditions on volatile substance changes and quality of Thompson seedless grape. Food and Fermentation Industries, 2015, 41(9): 181-187. (in Chinese)
[29]	PODOLYAN A, WHITE J, JORDAN B, WINEFIELD C. Identification of the lipoxygenase gene family from Vitis vinifera and biochemical characterisation of two 13-lipoxygenases expressed in grape berries of Sauvignon Blanc. Functional Plant Biology, 2010, 37(8): 767.
[30]	GARDNER H W. Recent investigations into the lipoxygenase pathway of plants. Biochimica et Biophysica Acta, 1991, 1084(3): 221-239. pmid: 1909580
[31]	KALUA C M, BOSS P K. Evolution of volatile compounds during the development of cabernet sauvignon grapes (Vitis vinifera L.). Journal of Agricultural and Food Chemistry, 2009, 57(9): 3818-3830.
[32]	DOLIGEZ A, BOUQUET A, DANGLOT Y, LAHOGUE F, RIAZ S, MEREDITH P, EDWARDS J, THIS P. Genetic mapping of grapevine (Vitis vinifera L.) applied to the detection of QTLs for seedlessness and berry weight. TAG Theoretical and Applied Genetics, 2002, 105(5): 780-795.
[33]	张海朋, 刘翠华, 刘园, 温欢, 施要强, 张红艳, 徐娟. 柑橘中挥发性萜类物质代谢研究进展. 园艺学报, 2020, 47(8): 1610-1624. doi: 10.16420/j.issn.0513-353x.2020-0100
	ZHANG H P, LIU C H, LIU Y, WEN H, SHI Y Q, ZHANG H Y, XU J. Research advances of volatile terpenoids metabolism in Citrus. Acta Horticulturae Sinica, 2020, 47(8): 1610-1624. (in Chinese)
[34]	JOSHI A, JEENA G S, SHIKHA, KUMAR R S, PANDEY A, SHUKLA R K. Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis. Plant Molecular Biology, 2022, 110(3): 235-251.
[35]	WANG N, SONG G W, ZHANG F J, SHU X C, CHENG G H, ZHUANG W B, WANG T, LI Y H, WANG Z. Characterization of the WRKY gene family related to anthocyanin biosynthesis and the regulation mechanism under drought stress and methyl jasmonate treatment in Lycoris radiata. International Journal of Molecular Sciences, 2023, 24(3): 2423.
[36]	SUN P W, XU Y H, YU C C, LV F F, TANG X L, GAO Z H, ZHANG Z, WANG H, LIU Y, WEI J H. WRKY44 represses expression of the wound-induced sesquiterpene biosynthetic gene ASS1 in Aquilaria sinensis. Journal of Experimental Botany, 2020, 71(3): 1128-1138.
[37]	ABBAS F, KE Y G, ZHOU Y W, YU Y Y, WASEEM M, ASHRAF U, LI X Y, YU R C, FAN Y P. Genome-wide analysis of ARF transcription factors reveals HcARF5 expression profile associated with the biosynthesis of β-ocimene synthase in Hedychium coronarium. Plant Cell Reports, 2021, 40(7): 1269-1284.
[38]	LOPEZ PINAR A, RAUHUT D, RUEHL E, BUETTNER A. Effects of bunch rot (Botrytis cinerea) and powdery mildew (Erysiphe necator) fungal diseases on wine aroma. Frontiers in Chemistry, 2017, 5: 20.
[39]	石莹, 刘园, 陈嘉景, 张海朋, 曾继吾, 黄宏建, 田静, 彭抒昂, 徐娟. 黄龙病病菌侵染对茶枝柑果实类黄酮和挥发性物质的影响. 华中农业大学学报, 2020, 39(1): 24-33.
	SHI Y, LIU Y, CHEN J J, ZHANG H P, ZENG J W, HUANG H J, TIAN J, PENG S A, XU J. Effects of Huanglongbing infection on profiles of flavonoids and volatiles in fruits of Citrus reticulata cv. Chachiensis. Journal of Huazhong Agricultural University, 2020, 39(1): 24-33. (in Chinese)

序号 Number	葡萄品种名称 Name of grape variety	属性 Attribute	果皮颜色 Skin color	类型 Type
1	无核白Thompson Seedless	欧亚Eurasian	白White	鲜食 Table grape
2	阳光玫瑰Shine Muscat	欧美Europe and America	金黄Golden yellow	鲜食 Table grape
3	红地球Red Globe	欧亚Eurasian	紫红Purplish red	鲜食Table grape
4	玫瑰香Muscat Hamburg	欧亚Eurasian	紫红Purplish red	鲜食Table grape
5	巨峰Kyoho	欧美Europe and America	紫黑Atropurpureus	鲜食Table grape
6	贵人香Italian Riesling	欧亚Eurasian	白White	酿酒Wine grape
7	小芒森Petit Manseng	欧亚Eurasian	白White	酿酒Wine grape
8	威代尔Vidal	欧美Europe and America	白White	酿酒Wine grape
9	赤霞珠Cabernet Sauvignon	欧亚Eurasian	紫黑Atropurpureus	酿酒Wine grape
10	蛇龙珠Cabernet Gernischet	欧亚Eurasian	紫黑Atropurpureus	酿酒Wine grape
11	梅鹿辄Merlot	欧亚Eurasian	紫黑Atropurpureus	酿酒 Wine grape
12	小味儿多Petit Verdot	欧亚Eurasian	紫黑Atropurpureus	酿酒 Wine grape
13	马瑟兰Marselan	欧亚Eurasian	深蓝Dark blue	酿酒 Wine grape

基因名称 Gene name	基因号 Gene ID	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
LOXA	VIT_06s0004g01510	TTTTCTTGGTTTAGGGATGA	CTTTGATTTGCCTTACTTCG
LOXC	VIT_14s0128g00780	TGAGCCTACCACATCCTAAT	TAGCCAGAGTCATTCACAGC
HPL	VIT_12s0059g01060	CCGTCCCACTCCAATACGCT	TTCGGGTCTGTCATCGCCAC
ADH1	VIT_18s0001g15410	AGCCATTAGTGATAGAAGAAGTG	GACATAGGGAGGTGTAGAGGA
LOXO	VIT_09s0002g01080	CTCAACGACCCACAACGCTAC	CGCACCTGTTTCTTCGGCTA
DXS	VIT_05s0020g02130	TTTGGTTGCGTCTTCTTTGC	GGACTCCACTTTGTTGTGCC

化学成分 Compound	阈值^[15,20] Threshold (μg∙L^-1)	香气特征^[15,20-21] Aromatic characteristic
正己醛 Hexanal	4.500	绿色蔬菜 Green vegetables
2-己烯醛 2-hexenal	17.000	青草，草本 Grass, herbaceous
反式-2-壬烯醛 Trans-2-Nonenal	0.080	潮湿的，土壤味 Moist, soil odor
(E,Z)-2,6-壬二烯醛 (E,Z)-2,6-nonadienal	0.020	紫罗兰和黄瓜香味 Violet and cucumber aromas
壬醛 Nonanal	1.000	脂肪，柑橘，青菜，果味 Fat, citrus, vegetables, fruity taste
异丁酸乙酯 Ethyl isobutyrate	0.100	果香 Fruity aroma
正己酸乙酯 Ethyl caproate	1.000	水果，青苹果，香蕉，葡萄酒，白兰地 Fruits, green apples, bananas, wine, brandy
辛酸乙酯 Ethyl octanoate	0.100	甜味，花香，水果味，香蕉，梨，白兰地 Sweet, floral, fruity, banana, pear, brandy
芳樟醇 Linalool	6.000	柑橘，花香，甜味 Citrus, floral aroma, sweet taste
香叶醇 Geraniol	10.000	柠檬味，花香，橙子味，玫瑰味 Lemon flavor, floral aroma, orange flavor, rose flavor
(E)-氧化玫瑰 (E)-Oxidized roses	0.100	玫瑰香，天竺兰香 Rose fragrance, geranium fragrance
β-紫罗兰酮 β-ionone	0.007	脂膏香，玫瑰香味，紫罗兰香 Cream fragrance, rose fragrance, violet fragrance

样品 Sample	原始数据 Raw reads	高质量数据 Clean reads	Q20 (%)	Q30 (%)	GC (%)
TS	7509114100	7377606868	97.63	93.23	46.38
SM	7984925800	7885274082	97.64	93.11	45.46
RG	6797327000	6629690349	97.57	93.21	46.49
MH	6863553200	6699407763	97.57	93.00	46.20
KY	6529118900	6378759691	97.83	93.82	46.20
IR	8716216500	8624849243	97.62	93.02	45.81
PM	6548941700	6424235773	97.72	93.61	46.40
VD	7417100700	7300033789	97.55	93.14	46.24
CS	7019247100	6845200698	97.39	92.48	46.67
CG	6581499100	6444723077	97.69	93.63	46.12
ML	7391406100	7271554342	97.56	93.24	46.43
PV	8206753300	8086178375	97.63	93.15	46.36
MS	7488201600	7299543929	97.36	92.31	46.20