红肉葡萄果实发育过程中花青苷组分变化与基因表达规律分析
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摘要
以红肉葡萄新种质‘钟山红玉’为实验材料,通过高效液相色谱-质谱联用(HPLC-MS)检测‘钟山红玉’从幼果到成熟期果皮、果肉中的花青苷组分及含量,并利用实时荧光定量PCR检测不同发育时期花青苷合成相关基因的表达水平,研究其不同果实发育时期果皮、果肉中的花青苷组分变化,以及相关基因表达规律,探索红肉葡萄果实呈色机制,为葡萄果实品质改良育种提供理论依据。结果表明:(1)‘钟山红玉’果皮和果肉中共检测出13种花青苷且都为单糖苷花青苷,与欧亚种葡萄亲缘关系较近。(2)‘钟山红玉’果皮、果肉中均检测出了欧亚种葡萄中很少有的天竺葵素3-O-葡萄糖苷;果皮中飞燕草素类(飞燕草素-、锦葵色素-、矮牵牛素-)花青苷衍生物含量显著高于矢车菊素类(矢车菊素-、芍药素-)花青苷衍生物,而在果肉中则两大类花青苷含量相近,这与果皮中较高的F3′5′H基因表达量有关,且在果皮中酰基化花青苷比例显著高于果肉。(3)花青苷合成相关基因MYBA2和UFGT在‘钟山红玉’不同发育时期的表达变化规律一致,UFGT基因在果肉中基本不表达,而MYBA2可能是决定‘钟山红玉’果肉转色的关键因子;并且ABA响应相关基因PYL1在‘钟山红玉’发育时期的表达规律与OMT和LDOX基因一致。
This study was conducted to explore the expression of the genes associated with anthocyanins biosynthesis and the differences of anthocyanins accumulation in the peel and pulp of one new red-flesh grape, ‘Zhongshanhongyu'(ZSHY). High-performance liquid chromatography with mass spectrometry(HPLC/MS) and qRT-PCR were used to understand the laws of anthocyanins accumulation and related genes expression during the berry development. It aims to provide a theoretical basis for improving quality of grapes. The results showed that:(1) there are total 13 kinds of anthocyanins were detected from the peel and pulp of ‘Zhongshanhongyu' and all of the anthocyanins were monoglucoside or monogalactoside. It is suggested that ‘ZSHY' probably belong to the cultivar Vitis vinifera.(2) One of detected anthocyanis, Pelargonidin-3-O-glucoside barely contained in the cultivar V. vinifera was detected in the peel and pulp of our material. Besides, the composition and content of anthocyanins was also various in different tissues. In the peel of ‘Zhongshanhongyu', the content of Delphinidin-based anthocyanins was significantly higher than Cyanidin-based anthocyanins, while the contents of Delphinidin-based and Cyanidin-based anthocyanins were almost same with each other. This result may due to the higher expression of F3′5′H in the peel of ‘Zhongshanhongyu'. In addition, the ratio of acylation anthocyanins was higher in the peel than in the pulp.(3) The regulation of the expression level of MYBA2 was consistent with UFGT, and UFGT barely expressed in the pulp, while MYBA2 maybe the key point that could regulate the pulp veraison. Besides, the expression of PYL1 which related to the regulation of ABA, also in according with the expression of OMT and LDOX.
This study was conducted to explore the expression of the genes associated with anthocyanins biosynthesis and the differences of anthocyanins accumulation in the peel and pulp of one new red-flesh grape, ‘Zhongshanhongyu'(ZSHY). High-performance liquid chromatography with mass spectrometry(HPLC/MS) and qRT-PCR were used to understand the laws of anthocyanins accumulation and related genes expression during the berry development. It aims to provide a theoretical basis for improving quality of grapes. The results showed that:(1) there are total 13 kinds of anthocyanins were detected from the peel and pulp of ‘Zhongshanhongyu' and all of the anthocyanins were monoglucoside or monogalactoside. It is suggested that ‘ZSHY' probably belong to the cultivar Vitis vinifera.(2) One of detected anthocyanis, Pelargonidin-3-O-glucoside barely contained in the cultivar V. vinifera was detected in the peel and pulp of our material. Besides, the composition and content of anthocyanins was also various in different tissues. In the peel of ‘Zhongshanhongyu', the content of Delphinidin-based anthocyanins was significantly higher than Cyanidin-based anthocyanins, while the contents of Delphinidin-based and Cyanidin-based anthocyanins were almost same with each other. This result may due to the higher expression of F3′5′H in the peel of ‘Zhongshanhongyu'. In addition, the ratio of acylation anthocyanins was higher in the peel than in the pulp.(3) The regulation of the expression level of MYBA2 was consistent with UFGT, and UFGT barely expressed in the pulp, while MYBA2 maybe the key point that could regulate the pulp veraison. Besides, the expression of PYL1 which related to the regulation of ABA, also in according with the expression of OMT and LDOX.
引文
[1] 高燕会,黄春红,朱玉球,等.植物花青素苷生物合成及调控的研究进展[J].中国生物工程杂志,2012,32(8):94-99.GAO Y H,HUANG C H,ZHU Y Q,et al .Progress on plant anthocyanin biosynthesis and regulation[J].China Biotechnology,2012,32(8):94-99.
[2] 李惠清,杨航宇,陈为凯,等.不同品种染色葡萄转色期花色苷积累的差异性研究[J].中国酿造,2018,37(9):140-147.LI H Q,YANG H Y,CHEN W K,et al.Difference of anthocyanins accumulation in different varieties of teinturier grapes at veraison[J].China Brewing,2018,37(9):140-147.
[3] AMIOT M J,RIVA C,VINET A.Effects of dietary polyphenols on metabolic syndrome features in humans:a systematic review[J].Obesity Reviews,2016,17(7):573-586.
[4] HOOPER L,KROON P A,RIMM E B,et al.Flavonoids,flavonoid-rich foods,and cardiovascular risk:a meta-analysis of randomized controlled trials[J].The American Journal of Clinical Nutrition,2008,88(1):38-50.
[5] KHOO H E,AZLAN A,TANG S T,et al.Anthocyanidins and anthocyanins:Colored pigments as food,pharmaceutical ingredients,and the potential health benefits[J].Food & Nutrition Research,2017,61(1):1 361 779.
[6] WANG N,ZHENG Y,DUAN N,et al.Comparative transcriptomes analysis of red-and white-fleshed apples in an F1 population of Malus sieversii f.Niedzwetzkyana crossed with M.domestica ‘Fuji’[J].PloS One,2015,10(7):e0133468.
[7] 罗静,谢汉忠,方金豹,等.红肉桃和白肉桃果实挥发性成分的差异分析[J].中国农业大学学报,2016,21(11):34-42.LUO J,XIE H Z,FANG J B,et al.Comparative analysis of volatile compounds in red-and white-fleshed peach[J].Journal of China Agricultural University,2016,21(11):34-42.
[8] LI W,LIU Y,ZENG S,et al.Gene expression profiling of development and anthocyanin accumulation in kiwifruit (Actinidia chinensis) based on transcriptome sequencing[J].PLoS One,2015,10(8):e0136439.
[9] MAZZA G,FRANCIS F J.Anthocyanins in grapes and grape products[J].Critical Reviews in Food Science & Nutrition,1995,35(4):341-371.
[10] JANVARY L,HOFFMANN T,PFEIFFER J,et al.A double mutation in the anthocyanin 5-O-glucosyltransferase gene disrupts enzymatic activity in Vitis vinifera L[J].Journal of Agricultural and Food Chemistry,2009,57(9):3 512-3 518.
[11] GARCíA-BENEYTEZ E,CABELLO F,REVILLA E.Analysis of grape and wine anthocyanins by HPLC-MS[J].Journal of Agricultural and Food Chemistry,2003,51(19):5 622-5 629.
[12] TANG K,LI Y,HAN Y,et al.Studies on the preparative isolation and stability of seven main anthocyanins from Yan 73 grape[J].Journal of the Science of Food and Agriculture,2014,94(12):2 472-2 481.
[13] MAZZA G,FUKUMOTO L,DELAQUIS P,et al.Anthocyanins,phenolics,and color of Cabernet franc,Merlot,and Pinot noir wines from British Columbia[J].Journal of Agricultural and Food Chemistry,1999,47(10):4 009-4 017.
[14] 王忠华,俞超,吴月燕.葡萄花青素苷生物合成研究进展[J].生物技术通报,2012,(9):1-7.WANG Z H,YU C,WU Y Y.Progress on anthocyanin biosynthesis in grape[J].Biotechnology Bulletin,2012,(9):1-7.
[15] 花庆,王蕾,韦灵林,等.矮牵牛F3′5′H基因的克隆及其在不同着色花药中的表达[J].西北农业学报,2011,20(8):152-158.HUA Q,WANG L,WEI L L,et al.Cloning and expression of F3′5′H gene in anther tissue with different colour in petunia hybrida[J].Acta Agriculturae Boreali-Occidentalis Sinica,2011,20(8):152-158.
[16] 邵建辉,马春花,祖艳群,等.转色初期喷施BTH对‘红地球’葡萄着色和果实品质的影响[J].果树学报,2017,34(12):1 556-1 565.SHAO J H,MA C H,ZU Y Q,et al.Effects of BTH application at veraison on berry coloration and quality of ‘Red Globe’ berries[J].Journal of Fruit Science,2017,34(12):1 556-1 565.
[17] 王华,李茂福,杨媛,等.果实花青素生物合成分子机制研究进展[J].植物生理学报,2015,51(1):29-43.WANG H,LI M F,YANG Y,et al.Recent advances on the molecular mechanisms of anthocyanin synthesis in fruits[J].Plant Physiology Journal,2015,51(1):29-43.
[18] KOBAYASHI S,ISHIMARU M,HIRAOKA K,et al.Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis[J].Planta,2002,215(6):924-933.
[19] DELUC L,BARRIEU F,MARCHIVE C,et al.Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway[J].Plant Physiology,2006,140(2):499-511.
[20] ALLAN A C,HELLENS R P,LAING W A.MYB transcription factors that colour our fruit[J].Trends in Plant Science,2008,13(3):99-102.
[21] HE F,LIANG N N,DUAN C Q,et al.Accumulation of anthocyanins in the skin and pulp of five different teinturier grapes[C]//IX International Symposium on Grapevine Physiology and Biotechnology,2013,1 157:73-82.
[22] CRUZ A A D L ,HILBERT G ,CéLINE RIVIèRE,et al.Anthocyanin identification and composition of wild Vitis spp.accessions by using LC-MS and LC-NMR[J].Analytica Chimica Acta,2012,732:145-152.
[23] BALIK J,KUM?TA M.Evaluation of colour content in grapes originating from South Moravia[J].Czech.J.Food Sci.,2008,26:18-24.
[24] BOSS P K,DAVIES C,ROBINSON S P.Anthocyanin composition and anthocyanin pathway gene expression in grapevine sports differing in berry skin colour[J].Australian Journal of Grape and Wine Research,1996,2(3):163-170.
[25] NIU T Q,GAO Z D,ZHANG P F,et al.MYBA2 gene involved in anthocyanin and flavonol biosynthesis pathways in grapevine[J].Genetics and Molecular Research,2016,15(4):gmr15048922.
[26] XIE S,ZHAO T,ZHANG Z,et al.Reduction of dihydrokaempferol by Vitis vinfera dihydroflavonol 4-reductase to produce orange pelargonidin-type anthocyanins[J].Journal of Agricultural and Food Chemistry,2018,66(13):3 524-3 532.
[27] MATSUYAMA S,TANZAWA F,KOBAYASHI H,et al.Leaf removal accelerated accumulation of delphinidin-based anthocyanins in ‘Muscat Bailey A’[Vitis×labruscana (Bailey) and Vitis vinifera (Muscat Hamburg)] grape skin[J].Journal of the Japanese Society for Horticultural Science,2013,83(1):17-22.
[28] HE J,GIUSTI M M.Anthocyanins:natural colorants with health-promoting properties[J].Annual Review of Food Science and Technology,2010,1:163-187.
[29] FALGINELLA L,DI GASPERO G,CASTELLARIN S D.Expression of flavonoid genes in the red grape berry of ‘Alicante Bouschet’varies with the histological distribution of anthocyanins and their chemical composition[J].Planta,2012,236(4):1 037-1 051.
[30] TARARA J M,LEE J,SPAYD S E,et al.Berry temperature and solar radiation alter acylation,proportion,and concentration of anthocyanin in Merlot grapes[J].American Journal of Enology and Viticulture,2008,59(3):235-247.
[31] 成果,周思泓,黄羽,等.‘桂葡 6 号’葡萄花色苷组成特点与基因表达规律解析[J].植物生理学报,2017,53(1):103-114.CHENG G,ZHOU S H,HUANG Y,et al.Analysis of anthocyanin composition characteristics and gene expression patterns of ‘Guipu No.6’ grape[J].Plant Physiology Journal,2017,53(1):103-114.
[32] HE J J,LIU Y X,PAN Q H,et al.Different anthocyanin profiles of the skin and the pulp of Yan73 (Muscat Hamburg×Alicante Bouschet) grape berries[J].Molecules,2010,15(3):1 141-1 153.
[33] JAAKOLA L.New insights into the regulation of anthocyanin biosynthesis in fruits[J].Trends in Plant Science,2013,18(9):477-483.
[34] SAVOI S,WONG D C J,DEGU A,et al.Multi-omics and integrated network analyses reveal new insights into the systems relationships between metabolites,structural genes,and transcriptional regulators in developing grape berries (Vitis vinifera L.) exposed to water deficit[J].Frontiers in Plant Science,2017,8:1 124.
[35] 孙欣,韩键,房经贵,等.葡萄浆果着色分子机理的重要研究进展[J].植物生理学报,2012,48(4):333-342.SUN X,HAN J,FANG J G,et al.Important research progress of coloring molecular mechanisms in grape berry[J].Plant Physiology Journal,2012,48(4):333-342.
[36] OGLESBY L,ANANGA A,OBUYA J,et al.Anthocyanin accumulation in muscadine berry skins is influenced by the expression of the MYB transcription factors,MybA1,and MYBCS1[J].Antioxidants,2016,5(4):35.
[37] JEONG S T,GOTO-YAMAMOTO N,HASHIZUME K,et al.Expression of the flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes and flavonoid composition in grape (Vitis vinifera)[J].Plant Science,2006,170(1):61-69.
[38] GAO Z,LI Q,LI J,et al.Characterization of the ABA receptor VlPYL1 that regulates anthocyanin accumulation in grape berry skin[J].Frontiers in Plant Science,2018,9:592.
[2] 李惠清,杨航宇,陈为凯,等.不同品种染色葡萄转色期花色苷积累的差异性研究[J].中国酿造,2018,37(9):140-147.LI H Q,YANG H Y,CHEN W K,et al.Difference of anthocyanins accumulation in different varieties of teinturier grapes at veraison[J].China Brewing,2018,37(9):140-147.
[3] AMIOT M J,RIVA C,VINET A.Effects of dietary polyphenols on metabolic syndrome features in humans:a systematic review[J].Obesity Reviews,2016,17(7):573-586.
[4] HOOPER L,KROON P A,RIMM E B,et al.Flavonoids,flavonoid-rich foods,and cardiovascular risk:a meta-analysis of randomized controlled trials[J].The American Journal of Clinical Nutrition,2008,88(1):38-50.
[5] KHOO H E,AZLAN A,TANG S T,et al.Anthocyanidins and anthocyanins:Colored pigments as food,pharmaceutical ingredients,and the potential health benefits[J].Food & Nutrition Research,2017,61(1):1 361 779.
[6] WANG N,ZHENG Y,DUAN N,et al.Comparative transcriptomes analysis of red-and white-fleshed apples in an F1 population of Malus sieversii f.Niedzwetzkyana crossed with M.domestica ‘Fuji’[J].PloS One,2015,10(7):e0133468.
[7] 罗静,谢汉忠,方金豹,等.红肉桃和白肉桃果实挥发性成分的差异分析[J].中国农业大学学报,2016,21(11):34-42.LUO J,XIE H Z,FANG J B,et al.Comparative analysis of volatile compounds in red-and white-fleshed peach[J].Journal of China Agricultural University,2016,21(11):34-42.
[8] LI W,LIU Y,ZENG S,et al.Gene expression profiling of development and anthocyanin accumulation in kiwifruit (Actinidia chinensis) based on transcriptome sequencing[J].PLoS One,2015,10(8):e0136439.
[9] MAZZA G,FRANCIS F J.Anthocyanins in grapes and grape products[J].Critical Reviews in Food Science & Nutrition,1995,35(4):341-371.
[10] JANVARY L,HOFFMANN T,PFEIFFER J,et al.A double mutation in the anthocyanin 5-O-glucosyltransferase gene disrupts enzymatic activity in Vitis vinifera L[J].Journal of Agricultural and Food Chemistry,2009,57(9):3 512-3 518.
[11] GARCíA-BENEYTEZ E,CABELLO F,REVILLA E.Analysis of grape and wine anthocyanins by HPLC-MS[J].Journal of Agricultural and Food Chemistry,2003,51(19):5 622-5 629.
[12] TANG K,LI Y,HAN Y,et al.Studies on the preparative isolation and stability of seven main anthocyanins from Yan 73 grape[J].Journal of the Science of Food and Agriculture,2014,94(12):2 472-2 481.
[13] MAZZA G,FUKUMOTO L,DELAQUIS P,et al.Anthocyanins,phenolics,and color of Cabernet franc,Merlot,and Pinot noir wines from British Columbia[J].Journal of Agricultural and Food Chemistry,1999,47(10):4 009-4 017.
[14] 王忠华,俞超,吴月燕.葡萄花青素苷生物合成研究进展[J].生物技术通报,2012,(9):1-7.WANG Z H,YU C,WU Y Y.Progress on anthocyanin biosynthesis in grape[J].Biotechnology Bulletin,2012,(9):1-7.
[15] 花庆,王蕾,韦灵林,等.矮牵牛F3′5′H基因的克隆及其在不同着色花药中的表达[J].西北农业学报,2011,20(8):152-158.HUA Q,WANG L,WEI L L,et al.Cloning and expression of F3′5′H gene in anther tissue with different colour in petunia hybrida[J].Acta Agriculturae Boreali-Occidentalis Sinica,2011,20(8):152-158.
[16] 邵建辉,马春花,祖艳群,等.转色初期喷施BTH对‘红地球’葡萄着色和果实品质的影响[J].果树学报,2017,34(12):1 556-1 565.SHAO J H,MA C H,ZU Y Q,et al.Effects of BTH application at veraison on berry coloration and quality of ‘Red Globe’ berries[J].Journal of Fruit Science,2017,34(12):1 556-1 565.
[17] 王华,李茂福,杨媛,等.果实花青素生物合成分子机制研究进展[J].植物生理学报,2015,51(1):29-43.WANG H,LI M F,YANG Y,et al.Recent advances on the molecular mechanisms of anthocyanin synthesis in fruits[J].Plant Physiology Journal,2015,51(1):29-43.
[18] KOBAYASHI S,ISHIMARU M,HIRAOKA K,et al.Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis[J].Planta,2002,215(6):924-933.
[19] DELUC L,BARRIEU F,MARCHIVE C,et al.Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway[J].Plant Physiology,2006,140(2):499-511.
[20] ALLAN A C,HELLENS R P,LAING W A.MYB transcription factors that colour our fruit[J].Trends in Plant Science,2008,13(3):99-102.
[21] HE F,LIANG N N,DUAN C Q,et al.Accumulation of anthocyanins in the skin and pulp of five different teinturier grapes[C]//IX International Symposium on Grapevine Physiology and Biotechnology,2013,1 157:73-82.
[22] CRUZ A A D L ,HILBERT G ,CéLINE RIVIèRE,et al.Anthocyanin identification and composition of wild Vitis spp.accessions by using LC-MS and LC-NMR[J].Analytica Chimica Acta,2012,732:145-152.
[23] BALIK J,KUM?TA M.Evaluation of colour content in grapes originating from South Moravia[J].Czech.J.Food Sci.,2008,26:18-24.
[24] BOSS P K,DAVIES C,ROBINSON S P.Anthocyanin composition and anthocyanin pathway gene expression in grapevine sports differing in berry skin colour[J].Australian Journal of Grape and Wine Research,1996,2(3):163-170.
[25] NIU T Q,GAO Z D,ZHANG P F,et al.MYBA2 gene involved in anthocyanin and flavonol biosynthesis pathways in grapevine[J].Genetics and Molecular Research,2016,15(4):gmr15048922.
[26] XIE S,ZHAO T,ZHANG Z,et al.Reduction of dihydrokaempferol by Vitis vinfera dihydroflavonol 4-reductase to produce orange pelargonidin-type anthocyanins[J].Journal of Agricultural and Food Chemistry,2018,66(13):3 524-3 532.
[27] MATSUYAMA S,TANZAWA F,KOBAYASHI H,et al.Leaf removal accelerated accumulation of delphinidin-based anthocyanins in ‘Muscat Bailey A’[Vitis×labruscana (Bailey) and Vitis vinifera (Muscat Hamburg)] grape skin[J].Journal of the Japanese Society for Horticultural Science,2013,83(1):17-22.
[28] HE J,GIUSTI M M.Anthocyanins:natural colorants with health-promoting properties[J].Annual Review of Food Science and Technology,2010,1:163-187.
[29] FALGINELLA L,DI GASPERO G,CASTELLARIN S D.Expression of flavonoid genes in the red grape berry of ‘Alicante Bouschet’varies with the histological distribution of anthocyanins and their chemical composition[J].Planta,2012,236(4):1 037-1 051.
[30] TARARA J M,LEE J,SPAYD S E,et al.Berry temperature and solar radiation alter acylation,proportion,and concentration of anthocyanin in Merlot grapes[J].American Journal of Enology and Viticulture,2008,59(3):235-247.
[31] 成果,周思泓,黄羽,等.‘桂葡 6 号’葡萄花色苷组成特点与基因表达规律解析[J].植物生理学报,2017,53(1):103-114.CHENG G,ZHOU S H,HUANG Y,et al.Analysis of anthocyanin composition characteristics and gene expression patterns of ‘Guipu No.6’ grape[J].Plant Physiology Journal,2017,53(1):103-114.
[32] HE J J,LIU Y X,PAN Q H,et al.Different anthocyanin profiles of the skin and the pulp of Yan73 (Muscat Hamburg×Alicante Bouschet) grape berries[J].Molecules,2010,15(3):1 141-1 153.
[33] JAAKOLA L.New insights into the regulation of anthocyanin biosynthesis in fruits[J].Trends in Plant Science,2013,18(9):477-483.
[34] SAVOI S,WONG D C J,DEGU A,et al.Multi-omics and integrated network analyses reveal new insights into the systems relationships between metabolites,structural genes,and transcriptional regulators in developing grape berries (Vitis vinifera L.) exposed to water deficit[J].Frontiers in Plant Science,2017,8:1 124.
[35] 孙欣,韩键,房经贵,等.葡萄浆果着色分子机理的重要研究进展[J].植物生理学报,2012,48(4):333-342.SUN X,HAN J,FANG J G,et al.Important research progress of coloring molecular mechanisms in grape berry[J].Plant Physiology Journal,2012,48(4):333-342.
[36] OGLESBY L,ANANGA A,OBUYA J,et al.Anthocyanin accumulation in muscadine berry skins is influenced by the expression of the MYB transcription factors,MybA1,and MYBCS1[J].Antioxidants,2016,5(4):35.
[37] JEONG S T,GOTO-YAMAMOTO N,HASHIZUME K,et al.Expression of the flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes and flavonoid composition in grape (Vitis vinifera)[J].Plant Science,2006,170(1):61-69.
[38] GAO Z,LI Q,LI J,et al.Characterization of the ABA receptor VlPYL1 that regulates anthocyanin accumulation in grape berry skin[J].Frontiers in Plant Science,2018,9:592.
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