不同颜色刺葡萄花色苷合成相关基因的表达分析
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摘要
【目的】探索不同果皮颜色中国野生刺葡萄(Vitis davidii)花色苷合成相关基因的表达差异,为中国野生刺葡萄资源的利用奠定基础。【方法】以白色及黑色果实的中国野生刺葡萄、欧亚种葡萄‘白比诺’及‘黑比诺’为材料,利用HPLC-ESI-MS/MS分析不同发育期果皮中花色苷的组成及其含量,通过序列检测分析几种刺葡萄Vvmy-bA1基因序列的不同,用实时荧光定量PCR检测花色苷合成相关结构基因表达的差异。【结果】锦葵色素葡萄糖苷(Malvidin 3-O-glucoside)仅在黑色果实转色后被检测到,其在中国野生刺葡萄黑色果实和‘黑比诺’中的含量分别为0.12～7.14mg/kg与4.90～180.79mg/kg,其他花色苷如矢车菊素3,5-O-双葡萄糖苷(Cyanidin 3-O-glucoside-5-O-glucosid)、飞燕草素3-O-葡萄糖苷(Delphinidin 3-O-glucoside)、花葵素3-O-葡萄糖苷(Pelargonidin 3-O-glucoside)、矮牵牛色素3-O-葡萄糖苷(Petunidin 3-O-glucosid)等在黑、白色葡萄果实不同发育期均能检测到,其含量为0.01～49.28mg/kg。VvmybA1a等位基因在中国野生刺葡萄黑、白色果实中都存在,且序列与欧亚种一致;VvmybA1c等位基因只在中国野生刺葡萄黑色果实中可检测到,与欧亚种相比其序列有33bp的插入片段和47bp的缺失片段。My-bA1基因只在黑色果实成熟期表达;合成花色苷的关键结构基因UFGT在供试葡萄各个时期均有表达,但其表达量在黑色果实成熟期显著高于白色果实;F3′5′H、GST及OMT等结构基因也在黑色果实成熟期表达上调,而其他结构基因的表达则在中国野生刺葡萄和欧亚种之间表现出了较大差异。【结论】中国野生刺葡萄花色苷合成相关基因具有特殊结构,与欧亚种葡萄相比有明显差异。
【Objective】This study investigated the differences in relative expression of anthocyanins biosynthesis structural genes between white skin berry and corresponding colored type of Chinese wild grape V.davidii to provide basis for its utilization in future.【Method】The compositions and contents of anthocyanins from four cultivars/lines including white skin berry and colored berry of V.davidii,‘Pinot Blanc'and‘Pinot Noir'of V.vinifera were analyzed by HPLC-ESI-MS/MS.The MybA1 transcription factors regulating anthocyanin biosynthesis were detected,and the gene sequences of MybA1 were cloned from different samples and compared to explore the relationships between Vitis species.The expressions of anthocyanins related biosynthesis structural genes were analyzed by real-time PCR.【Result】Only malvidin 3-Oglucoside was detected in color skin grapes during ripening stage,and the contents of colored V.davidii and‘Pinot Noir'were 0.12-7.14 mg/kg and 4.90-180.79 mg/kg,respectively.The contents of other anthocyanins(0.01-49.28 mg/kg),such as cyanidin 3-O-glucoside-5-O-glucosid,delphinidin 3-O-glucoside,pelargonidin 3-O-glucoside and petunidin 3-O-glucosid,were lower by several-fold to hundreds-fold compared to the colored grape varieties.Compared with V.vinifera,there were a 33 bp short insertion and a 47 bp gene fragment missing in the VvmybA1 c allele of V.davidii.MybA1 regulated anthocyanin accumulation in grape berry skin via UFGT gene expression.Colored berry skin had high levels of MybA1 and UFGT genes expression at harvest stages compared with white berry skin.In addition,the levels of F3′5′H,GSTand OMTgenes expression were similar to UFGT.Other structure genes expressions were significantly different between V.davidii and V.vinifera.【Conclusion】There were significant differences identified in the sequences of MybA1 between V.viniferaand V.davidii.
【Objective】This study investigated the differences in relative expression of anthocyanins biosynthesis structural genes between white skin berry and corresponding colored type of Chinese wild grape V.davidii to provide basis for its utilization in future.【Method】The compositions and contents of anthocyanins from four cultivars/lines including white skin berry and colored berry of V.davidii,‘Pinot Blanc'and‘Pinot Noir'of V.vinifera were analyzed by HPLC-ESI-MS/MS.The MybA1 transcription factors regulating anthocyanin biosynthesis were detected,and the gene sequences of MybA1 were cloned from different samples and compared to explore the relationships between Vitis species.The expressions of anthocyanins related biosynthesis structural genes were analyzed by real-time PCR.【Result】Only malvidin 3-Oglucoside was detected in color skin grapes during ripening stage,and the contents of colored V.davidii and‘Pinot Noir'were 0.12-7.14 mg/kg and 4.90-180.79 mg/kg,respectively.The contents of other anthocyanins(0.01-49.28 mg/kg),such as cyanidin 3-O-glucoside-5-O-glucosid,delphinidin 3-O-glucoside,pelargonidin 3-O-glucoside and petunidin 3-O-glucosid,were lower by several-fold to hundreds-fold compared to the colored grape varieties.Compared with V.vinifera,there were a 33 bp short insertion and a 47 bp gene fragment missing in the VvmybA1 c allele of V.davidii.MybA1 regulated anthocyanin accumulation in grape berry skin via UFGT gene expression.Colored berry skin had high levels of MybA1 and UFGT genes expression at harvest stages compared with white berry skin.In addition,the levels of F3′5′H,GSTand OMTgenes expression were similar to UFGT.Other structure genes expressions were significantly different between V.davidii and V.vinifera.【Conclusion】There were significant differences identified in the sequences of MybA1 between V.viniferaand V.davidii.
引文
[1]刘崇怀,姜建福,樊秀彩,等.中国野生葡萄资源在生产和育种中利用的概况[J].植物遗传资源学报,2014,15(4):720-727.Liu C H,Jiang J F,Fan X C,et al.The utilization of Chinese wild grape species in production and breeding[J].Journal of Plant Genetic Resources,2014,15(4):720-727.
[2]石雪晖,杨国顺,熊兴耀.湖南省刺葡萄种质资源的研究与利用[J].湖南农业科学,2010(19):1-4.Shi X H,Yang G S,Xiong X Y.Research and utilization status of germplasm resources of Vitis davidii in Hunan[J].Hunan Agricultural Sciences,2010(19):1-4.
[3]Jiao J,Fu X,Liu C,et al.Study of the relationship between the cultivars of Vitis vinifera and the white-fruited and hermaphrodite Chinese wild grapes[J].Molecular Breeding,2014,34(3):1401-1411.
[4]Mamiko S,Keiko F,Hironori K,et al.Pink-colored grape berry is the result of short insertion in intron of color regulatory gene[J].PLoS One,2011,66(6):21308-21318.
[5]Kobayashi S,Goto-Yamamoto N,Hirochika H.Retrotransposon-induced mutations in grape skin color[J].Science,2004,304:982.
[6]Cutanda-Perez M C,Ageorges A,Gomez C,et al.Ectopic expression of VlmybA1in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport[J].Plant Molecular Biology,2008,69(6):633-648.
[7]Czemmel S,Stracke R,Weisshaar B,et al.The grapevine R2R3-MYB transcription factor VvMYBF1regulates flavonol synthesis in developing grape berries[J].Plant Physiology,2009,151(3):1513-1530.
[8]Mitani N,Azuma A,Hirochika H,et al.A retrotransposon-inserted VvmybA1aallele has been spread among cultivars of Vitis vinifera but not North American or East Asian vitis species[J].Vitis,2009,48(1):55-56.
[9]This P,Lacombe T,Cadle-Davidson M,et al.Wine grape(Vitis vinifera L.)color associates with allelic variation in the domestication gene VvmybA1[J].Theoretical and Applied Genetics,2007,114(4):723-730.
[10]慕茜,吴为民,房经贵,等.不同葡萄品种的VvmybA1基因型及其特征性DNA片段的序列分析[J].园艺学报,2011,38(11):2075-2084.Mu Q,Wu W M,Fang J G,et al.Genotyping VvmybA1in 59grapevine cultivars and characterization of the DNA fragement sequences acta[J].Horticulturae Sinica,2011,38(11):2075-2084.
[11]Dietmar K,Achim C,Reinhold C,et al.Polyphenol screening of pomace from red and white grape varieties(Vitis vinifera L.)by HPLC-DAD-MS/MS[J].Journal of Agricultural and Food Chemistry,2004,52:4360-4367.
[12]Liang Z,Owens C L,Zhong G Y,et al.Polyphenolic profiles detected in the ripe berries of Vitis vinifera germplasm[J].Food Chemistry,2011,129(3):940-950.
[13]Arapitsas P,Oliveira J,Mattivi F.Do white grapes really exist?[J].Food Research International,2015,69:21-25.
[14]Yakushiji H,Kobayashi S,Goto-Yamamoto N,et al.A skin color mutation of grapevine,from black-skinned Pinot noir to whiteskinned Pinot blanc,is caused by deletion of the functional VvmybA1allele[J].Bioscience,Biotechnology,and Biochemistry,2006,70(6):1506-1508.
[15]Kobayashi S,Goto-Yamamoto N,Hirochika H.Association of VvmybA1gene expression with anthocyanin production in grape(Vitis vinifera)skin-color mutants[J].Journal of Japan Soc Hort Science,2005,74(3):196-203.
[16]Azuma A,Kobayashi S,Mitani N,et al.Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin[J].Theoretical and Applied Genetics,2008,117(6):1009-1019.
[17]Jeong S T,Goto-Yamamoto N,Kobayashi S,et al.Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins[J].Plant Science,2004,167(2):247-252.
[18]Reid K E,Olsson N,Schlosser J,et al.An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[J].BMC Plant Biology,2006,6(1):27-38.
[19]Schmittgen T D,Livak K J.Analyzing real-time PCR data by the comparative CT method[J].Nature Protocols,2008,3(6):1101-1108.
[20]Liang Z,Wu B,Fan P,et al.Anthocyanin composition and content in grape berry skin in Vitis germplasm[J].Food Chemistry,2008,111(4):837-844.
[21]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):1141-1153.
[22]Castellarin S D,Gaspero G D,Macconi R.Colour variation in red grapevines(Vitis vinifera L.):genomic organization,expression of flavonoid 3′-hydroxylase,flavonoid 3′,5′-hydroxylase genes and related metabolite profiling of red cyanidingblue delphinidin-based anthocyanins in berry skin[J].BMCGenomics,2006,7:12.
[23]Jeong S T,Goto-Yamaoto N,Hashizume K.Expression of the flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxlase genes and flavonoid composition in grapes and flavonoid composition in grape(Vitis vinifera L.)[J].Plant Science,2006,170:61-69.
[24]Lijavetzky D,Ruiz-García L,Cabezas J A,et al.Molecular genetics of berry colour variation in table grape[J].Molecular Genetics and Genomics,2006,276(5):427-435.
[25]Bogs J.Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development[J].Plant Physiology,2005,140(1):279-291.
[26]Prior R L,Wu X L.Anthocyanins:structural characteristics that result in unique metabolic patterns and biological activities[J].Free Radical Research,2006,40(10):1014-1028.
[27]Szajdek A,Borowska E J.Bioactive compounds and health-promoting properties of berry fruits:a review[J].Plant Foods for Human Nutrition,2008,63(4):147-156.
[28]Wu X,Prior R L.Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States:fruits and berries[J].Journal of Agricultural and Food Chemistry,2005,53:2589-2599.
[29]Haselgrove L,Botting D,Heeswijck R,et al.Canopy microclimate and berry composition the effect of bunch exposure on the phenolic composition of Vitis vinifera L.cv.Shiraz grape berries[J].Australian Journal of Grape and Wine Research,2000,6:141-149.
[30]Esteban M A,Villanueva M J,Lissarrague J R.Effect of irrigation on changes in the anthocyanin composition of the skin of cv Tempranillo(Vitis vinifera L.)grape berries during ripening[J].Journal of the Science of Food and Agriculture,2001,81:409-420.
[31]Ryan J M,Revilla E.Anthocyanin composition of Cabernet Sauvignon and Tempranillo grapes at different stages of ripening[J].Journal of Agricultural and Food Chemistry,2003,51:3372-3378.
[32]Yamane T,Jeong S T,Goto-Yamamoto N,et al.Effects of temperature on anthocyanin biosynthesis in grape berry skins[J].American Journal of Enology and Viticulture,2006,57:54-59.
[33]Laatti A K,Kainulainen P S,Hayirlioglu-Ayaz S,et al.Characterization of anthocyanins in caucasian blueberries(Vaccinium arctostaphylos L.)native to Turkey[J].Journal of Agricultural and Food Chemistry,2009,57(12):5244-5249.
[34]Arapitsas P,Perenzoni D,Nicolini G,et al.Study of sangiovese wines pigment profile by UHPLC-MS/MS[J].Journal of Agricultural and Food Chemistry,2012,60(42):10461-10471.
[2]石雪晖,杨国顺,熊兴耀.湖南省刺葡萄种质资源的研究与利用[J].湖南农业科学,2010(19):1-4.Shi X H,Yang G S,Xiong X Y.Research and utilization status of germplasm resources of Vitis davidii in Hunan[J].Hunan Agricultural Sciences,2010(19):1-4.
[3]Jiao J,Fu X,Liu C,et al.Study of the relationship between the cultivars of Vitis vinifera and the white-fruited and hermaphrodite Chinese wild grapes[J].Molecular Breeding,2014,34(3):1401-1411.
[4]Mamiko S,Keiko F,Hironori K,et al.Pink-colored grape berry is the result of short insertion in intron of color regulatory gene[J].PLoS One,2011,66(6):21308-21318.
[5]Kobayashi S,Goto-Yamamoto N,Hirochika H.Retrotransposon-induced mutations in grape skin color[J].Science,2004,304:982.
[6]Cutanda-Perez M C,Ageorges A,Gomez C,et al.Ectopic expression of VlmybA1in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport[J].Plant Molecular Biology,2008,69(6):633-648.
[7]Czemmel S,Stracke R,Weisshaar B,et al.The grapevine R2R3-MYB transcription factor VvMYBF1regulates flavonol synthesis in developing grape berries[J].Plant Physiology,2009,151(3):1513-1530.
[8]Mitani N,Azuma A,Hirochika H,et al.A retrotransposon-inserted VvmybA1aallele has been spread among cultivars of Vitis vinifera but not North American or East Asian vitis species[J].Vitis,2009,48(1):55-56.
[9]This P,Lacombe T,Cadle-Davidson M,et al.Wine grape(Vitis vinifera L.)color associates with allelic variation in the domestication gene VvmybA1[J].Theoretical and Applied Genetics,2007,114(4):723-730.
[10]慕茜,吴为民,房经贵,等.不同葡萄品种的VvmybA1基因型及其特征性DNA片段的序列分析[J].园艺学报,2011,38(11):2075-2084.Mu Q,Wu W M,Fang J G,et al.Genotyping VvmybA1in 59grapevine cultivars and characterization of the DNA fragement sequences acta[J].Horticulturae Sinica,2011,38(11):2075-2084.
[11]Dietmar K,Achim C,Reinhold C,et al.Polyphenol screening of pomace from red and white grape varieties(Vitis vinifera L.)by HPLC-DAD-MS/MS[J].Journal of Agricultural and Food Chemistry,2004,52:4360-4367.
[12]Liang Z,Owens C L,Zhong G Y,et al.Polyphenolic profiles detected in the ripe berries of Vitis vinifera germplasm[J].Food Chemistry,2011,129(3):940-950.
[13]Arapitsas P,Oliveira J,Mattivi F.Do white grapes really exist?[J].Food Research International,2015,69:21-25.
[14]Yakushiji H,Kobayashi S,Goto-Yamamoto N,et al.A skin color mutation of grapevine,from black-skinned Pinot noir to whiteskinned Pinot blanc,is caused by deletion of the functional VvmybA1allele[J].Bioscience,Biotechnology,and Biochemistry,2006,70(6):1506-1508.
[15]Kobayashi S,Goto-Yamamoto N,Hirochika H.Association of VvmybA1gene expression with anthocyanin production in grape(Vitis vinifera)skin-color mutants[J].Journal of Japan Soc Hort Science,2005,74(3):196-203.
[16]Azuma A,Kobayashi S,Mitani N,et al.Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin[J].Theoretical and Applied Genetics,2008,117(6):1009-1019.
[17]Jeong S T,Goto-Yamamoto N,Kobayashi S,et al.Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins[J].Plant Science,2004,167(2):247-252.
[18]Reid K E,Olsson N,Schlosser J,et al.An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[J].BMC Plant Biology,2006,6(1):27-38.
[19]Schmittgen T D,Livak K J.Analyzing real-time PCR data by the comparative CT method[J].Nature Protocols,2008,3(6):1101-1108.
[20]Liang Z,Wu B,Fan P,et al.Anthocyanin composition and content in grape berry skin in Vitis germplasm[J].Food Chemistry,2008,111(4):837-844.
[21]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):1141-1153.
[22]Castellarin S D,Gaspero G D,Macconi R.Colour variation in red grapevines(Vitis vinifera L.):genomic organization,expression of flavonoid 3′-hydroxylase,flavonoid 3′,5′-hydroxylase genes and related metabolite profiling of red cyanidingblue delphinidin-based anthocyanins in berry skin[J].BMCGenomics,2006,7:12.
[23]Jeong S T,Goto-Yamaoto N,Hashizume K.Expression of the flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxlase genes and flavonoid composition in grapes and flavonoid composition in grape(Vitis vinifera L.)[J].Plant Science,2006,170:61-69.
[24]Lijavetzky D,Ruiz-García L,Cabezas J A,et al.Molecular genetics of berry colour variation in table grape[J].Molecular Genetics and Genomics,2006,276(5):427-435.
[25]Bogs J.Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development[J].Plant Physiology,2005,140(1):279-291.
[26]Prior R L,Wu X L.Anthocyanins:structural characteristics that result in unique metabolic patterns and biological activities[J].Free Radical Research,2006,40(10):1014-1028.
[27]Szajdek A,Borowska E J.Bioactive compounds and health-promoting properties of berry fruits:a review[J].Plant Foods for Human Nutrition,2008,63(4):147-156.
[28]Wu X,Prior R L.Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States:fruits and berries[J].Journal of Agricultural and Food Chemistry,2005,53:2589-2599.
[29]Haselgrove L,Botting D,Heeswijck R,et al.Canopy microclimate and berry composition the effect of bunch exposure on the phenolic composition of Vitis vinifera L.cv.Shiraz grape berries[J].Australian Journal of Grape and Wine Research,2000,6:141-149.
[30]Esteban M A,Villanueva M J,Lissarrague J R.Effect of irrigation on changes in the anthocyanin composition of the skin of cv Tempranillo(Vitis vinifera L.)grape berries during ripening[J].Journal of the Science of Food and Agriculture,2001,81:409-420.
[31]Ryan J M,Revilla E.Anthocyanin composition of Cabernet Sauvignon and Tempranillo grapes at different stages of ripening[J].Journal of Agricultural and Food Chemistry,2003,51:3372-3378.
[32]Yamane T,Jeong S T,Goto-Yamamoto N,et al.Effects of temperature on anthocyanin biosynthesis in grape berry skins[J].American Journal of Enology and Viticulture,2006,57:54-59.
[33]Laatti A K,Kainulainen P S,Hayirlioglu-Ayaz S,et al.Characterization of anthocyanins in caucasian blueberries(Vaccinium arctostaphylos L.)native to Turkey[J].Journal of Agricultural and Food Chemistry,2009,57(12):5244-5249.
[34]Arapitsas P,Perenzoni D,Nicolini G,et al.Study of sangiovese wines pigment profile by UHPLC-MS/MS[J].Journal of Agricultural and Food Chemistry,2012,60(42):10461-10471.