葡萄着色期果皮蛋白质组分析
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摘要
为了研究葡萄不同着色期果皮中蛋白质表达特征,以着色初期、中期和后期等3个着色时期的葡萄果皮为研究对象,运用2-DE、MALDI-TOF/TOF-MS质谱技术以及生物信息学方法对差异蛋白进行分析,结果表明:(1)双向凝胶电泳显示,果皮着色3个时期有1050个高度重复的蛋白点,差异显著的蛋白点有162个,其中108个蛋白得到鉴定,有87个蛋白映射到葡萄蛋白质组数据库中;3个时期均表达的差异蛋白有20个,且随着果皮颜色加深,差异蛋白数量呈上升趋势。(2)GO分析显示,ATP合成酶β亚基(ATPβ)极显著富集于磷酸核糖代谢过程,对着色初期果皮生理代谢的能量需求具有重要意义。(3)KEGG分析表明,碳代谢、生物固碳、磷酸戊糖、氨基酸生物合成等代谢通路在果皮着色的不同时期显著富集。(4)qRT-PCR分析显示,S–腺苷甲硫氨酸合成酶基因(VvMETK4)在果皮着色后期表达量最高。
In order to study protein expression characteristics of grape pericarp in different coloring periods,early,middle and later stages of pericarp coloring were studied. 2-DE(Two-dimensional gel electrophoresis)and MALDI-TOF/TOF-MS(mass spectrometry technology)was used to analyze the differences of protein expression in pericarp at different coloring stages.(1) 2-DE revealed that there were1 050 highly repeated protein spots during 3 periods of pericarp coloring,and 108 of 162 differentialexpressed proteins were identified,87 proteins were mapped to the grape database. Twenty proteins differentially expressed during 3 periods of pericarp coloring,and the number of differential-expressed proteins was increasing with the deepening process of pericarp color.(2) GO enrichment analysis showed ATPβ(ATPase β subunit)was highly enriched in the ribose phosphate metabolic process,which was of great significance to the energy requirement of physiological metabolism in the early stage of coloring.(3)KEGG analysis showed that many KEGG pathways,such as carbon metabolism,photosynthetic carbon fixation,pentose phosphate,amino acid biosynthesis,metabolic pathways etc,were significantly enriched at different stages of pericarp coloration.(4) qRT-PCR analysis showed that the S-adenosylmethionine synthetase 4 gene(VvMETK4)had the highest expression level in the later stage of pericarp coloring.
In order to study protein expression characteristics of grape pericarp in different coloring periods,early,middle and later stages of pericarp coloring were studied. 2-DE(Two-dimensional gel electrophoresis)and MALDI-TOF/TOF-MS(mass spectrometry technology)was used to analyze the differences of protein expression in pericarp at different coloring stages.(1) 2-DE revealed that there were1 050 highly repeated protein spots during 3 periods of pericarp coloring,and 108 of 162 differentialexpressed proteins were identified,87 proteins were mapped to the grape database. Twenty proteins differentially expressed during 3 periods of pericarp coloring,and the number of differential-expressed proteins was increasing with the deepening process of pericarp color.(2) GO enrichment analysis showed ATPβ(ATPase β subunit)was highly enriched in the ribose phosphate metabolic process,which was of great significance to the energy requirement of physiological metabolism in the early stage of coloring.(3)KEGG analysis showed that many KEGG pathways,such as carbon metabolism,photosynthetic carbon fixation,pentose phosphate,amino acid biosynthesis,metabolic pathways etc,were significantly enriched at different stages of pericarp coloration.(4) qRT-PCR analysis showed that the S-adenosylmethionine synthetase 4 gene(VvMETK4)had the highest expression level in the later stage of pericarp coloring.
引文
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Claeyssen E,Rivoal J.2007.Isozymes of plant hexokinase:occurrence,properties and functions.Phytochemistry,68(6):709-731.
Coulthurst S J,Lilley K S,Hedley P E,Liu H,Toth I K,Salmond G P.2008.DsbA plays a critical and multi-faceted role in the production of secreted virulence factors by the phytopathogen Erwinia carotovora subsp.atroseptica.J Biol Chem,283(35):23739-23753.
Damerval C,De Vienne D,Zivy M,Thiellement H.1986.Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins.Electrophoresis,7:52-54.
Devaiah Kambiranda,Ramesh Katam,Sheikh M Basha,Shalom Siebert.2014.iTRAQ-based quantitative proteomics of developing and ripening muscadine grape berry.J Proteome Res,13:555-569.
Di Carli M,Zamboni A,PèM E,Pezzotti M,Lilley K S,Benvenuto E,Desiderio A.2011.Two-dimensional differential in gel electrophoresis(2D-DIGE)analysis of grape berry proteome during postharvest withering.Journal of Proteome Research,10:429-446.
El-Kereamy A,Chervin C,Roustan J P,Cheynier V,Souquet J M,Moutounet M,Raynal J,Ford C,Latche A,Pech J C,Bouzayen M.2003.Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries.Physiologia Plantarum,119:175-182.
Feniouk B A,Suzuki T,Yoshida M.2006.The role of subunit epsilon in the catalysis and regulation of FoF1-ATP synthase.Biochim Biophysica Acta,1757(5-6):326-338.
Fernández-Oca?a A,Chaki M,Luque F,Gómez-Rodríguez M V,Carreras A,Valderrama R,Begara-Morales J C,Hernández L E,Corpas F J,Barroso J B.2011.Functional analysis of superoxide dismutases(SODs)in sunflower under biotic and abiotic stress conditions.Identification of two new genes of mitochondrial Mn-SOD.J Plant Physiol,168(11):1303-1308.
Guo Shao-jian.2016.Analysis of transcription and protein expression of VvHY5 and VvCOP1 in grape peel under light removal condition[Ph.D.Dissertation].Wuhan:Graduate University of Chinese Academy of Sciences.(in Chinese)郭少剑.2016.去光条件下葡萄果皮中VvHY5和VvCOP1的转录和蛋白表达分析[博士论文].武汉:中国科学院研究生院.
Gutteridge S,Gatenby A A.1995.Rubisco synthesis,assembly,mechanism,and regulation.Plant Cell,7(7):809-819.
Huang Yao,Ma Cheng,Wu Nai-hu,Li Zhao-luan.1994.Structural analysis of grape chloroplast rbcL gene.Acta Botanica Sinica,36(6):444-451.(in Chinese)黄瑶,马诚,吴乃虎,李朝銮.1994.葡萄叶绿体rbcL基因的结构分析.植物学报,36(6):444-451.
Jellouli N,Ben Jouira H,Skouri H,Ghorbel A,Gourgouri A,Mliki A.2008.Proteomic analysis of Tunisian grapevine cultivar Razegui under salt stress.J Plant Physiol,165:471-481.
Jin Liang,Chen Shang-wu,Ma Hui-qin.2010.Advances in grape proteomics.China Biotechnology,30(10):100-107.(in Chinese)金良,陈尚武,马会勤.2010.葡萄蛋白质组学研究进展.中国生物工程杂志,30(10):100-107.
Li Feng.2011.Construction and EST analysis of cDNA library from mature peel of Vitis amurensis Rupr[M.D.Dissertation].Harbin:Northeast Forestry University.(in Chinese)李凤.2011.山葡萄成熟果皮cDNA文库的构建及EST分析[硕士论文].哈尔滨:东北林业大学.
Li Ming-liang,Han Yi-fan.2000.Effect of ethylene on the growth and development of plants and inhibition of its biosynthesis by antisense RNA.Seientia Silvae Sinicae,36(4):77-84.(in Chinese)李明亮,韩一凡.2000.乙烯在植物生长发育的抗病反应中的作用及其生物合成的反义抑制.林业科学,36(4):77-84.
Liu G T,Chai F M,Wang Y,Jiang J Z,Duan W,Wang Y T,Wang F F,Li S H,Wang L J.2018.Genome-wide identification and classification of HSF family in grape,and their transcriptional analysis under heat acclimation and heat stress.Horticultural Plant Journal,4(4):133-143.
Mei Yang,Li Hai-lan,Xie Jin,Luo Hong-yi.2007.Ribose-1,5-diphosphate carboxylase/oxygenase(Rubisco).Plant Physiology Communications,(2):363-368.(in Chinese)梅杨,李海蓝,谢晋,罗红艺.2007.核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco).植物生理学通讯,(2):363-368.
Merali S,Boykin C A.2004.S-adenosylmethionine and Pneu-mocystis.FEMS Microbiol Lett,237(1):179-186.
Niu Jiao.2012.Cloning and expression analysis of response genes of powdery mildew of wild Vitis vinifera in China[Ph.D.Dissertation].Yangling:Northwest A&F University.(in Chinese)牛姣.2012.中国野生毛葡萄白粉病响应基因克隆及表达分析[博士论文].杨凌:西北农林科技大学.
Shibli R A,Smith M A L,Kushad M.1997.Headspace ethylene accumulation effects on secondary metabolite production in Vaccinium pahalae cell culture.Plant Growth Reg,23:201-205.
Shimizu T,Tanizawa Y,Mochizuki T,Nagasaki H,Yoshioka T,Toyoda A,Fujiyama A,Kaminuma E,Nakamura Y.2017.Draft Sequencing of the heterozygous diploid genome of satsuma(Citrus unshiu Marc.)Using a Hybrid Assembly Approach Front Genet,8:180.
Spagnolo A,Magnin-Robert M,Alayi T D,Cilindre C,Mercier L,Schaeffer-Reiss C,van Dorsselaer A,Clément C,Fontaine F.2012.Physiological changes in green stems of Vitis vinifera L.cv.Chardonnay in response to esca proper and apoplexy revealed by proteomic and transcriptomic analyses.J Proteome Res,11:461-475.
Wu Jun-lin,Wu Qing-ping,Zhang Ju-mei,Zhang Wen.2015.Progress in L-malic acid biosynthesis.The Food Industry,36(9):225-228.(in Chinese)吴军林,吴清平,张菊梅,张文.2015.L-苹果酸的生物学功能研究新进展.食品工业,36(9):225-228.
Xu Fan.2015.Effect of Brassinolide on sugar unloading during grape maturation[Ph.D.Dissertation].Yangling:Northwest A&F University.(in Chinese)徐凡.2015.油菜素内酯对葡萄成熟过程中糖卸载的影响[博士论文].杨凌:西北农林科技大学.
Yang K A,Moon H J,Kim G T,Ju L C,Hong J C,Oh L C,Yun D J.2003.NDP kinase 2 regulates expression of antioxidant genes in Arabidopsis.Proc Jpn Acad Ser B,79(1):86-91.
Yang Ying-ying,Gao Yang,Zheng Jia-peng,Gan Wu,Xiang Miao-lian,Chen Ming,Chen Jin-yin.2016.Adcances in research on the approach of degradation metabolism of citric acid in citrus fruit and it’s regulation.Acta Agriculturae Universitatis Jiangxinsis,38(3):476-482.(in Chinese)杨滢滢,高阳,郑嘉鹏,甘武,向妙莲,陈明,陈金印.2016.柑橘果实柠檬酸降解代谢途径与调控研究进展.江西农业大学学报,38(3):476-482.
Zhao Jin,Sun Mei-hong,Hu Da-gang,Hao Yu-jin.2015.Molecular cloning and expression analysis of a hexokinase gene MdHXK1 in apple.Acta Horticulturae Sinica,42(8):1437-1447.(in Chinese)赵锦,孙美红,胡大刚,郝玉金.2015.苹果己糖激酶基因MdHXK1的克隆与表达分析.园艺学报,42(8):1437-1447.
Zhang Wen-li,Shen Wen-biao,Ye Mao-bing,Xu Lang-lai.2003.Plant cis and its physiological functions.Plant Physiology Communications,(4):391-398.(in Chinese)张文利,沈文飚,叶茂炳,徐朗莱.2003.植物顺乌头酸酶及其生理功能.植物生理学通讯,(4):391-398.
Chen Y Y,Zhang Z H,Zhong C Y,Song X M,Lin Q H,Huang C M,Huang R H,Chen W.2016.Functional analysis of differentially expressed proteins in Chinese bayberry(Myrica rubra Sieb.et Zucc.)fruits during ripening.Food Chemistry,190:763-770.
Claeyssen E,Rivoal J.2007.Isozymes of plant hexokinase:occurrence,properties and functions.Phytochemistry,68(6):709-731.
Coulthurst S J,Lilley K S,Hedley P E,Liu H,Toth I K,Salmond G P.2008.DsbA plays a critical and multi-faceted role in the production of secreted virulence factors by the phytopathogen Erwinia carotovora subsp.atroseptica.J Biol Chem,283(35):23739-23753.
Damerval C,De Vienne D,Zivy M,Thiellement H.1986.Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins.Electrophoresis,7:52-54.
Devaiah Kambiranda,Ramesh Katam,Sheikh M Basha,Shalom Siebert.2014.iTRAQ-based quantitative proteomics of developing and ripening muscadine grape berry.J Proteome Res,13:555-569.
Di Carli M,Zamboni A,PèM E,Pezzotti M,Lilley K S,Benvenuto E,Desiderio A.2011.Two-dimensional differential in gel electrophoresis(2D-DIGE)analysis of grape berry proteome during postharvest withering.Journal of Proteome Research,10:429-446.
El-Kereamy A,Chervin C,Roustan J P,Cheynier V,Souquet J M,Moutounet M,Raynal J,Ford C,Latche A,Pech J C,Bouzayen M.2003.Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries.Physiologia Plantarum,119:175-182.
Feniouk B A,Suzuki T,Yoshida M.2006.The role of subunit epsilon in the catalysis and regulation of FoF1-ATP synthase.Biochim Biophysica Acta,1757(5-6):326-338.
Fernández-Oca?a A,Chaki M,Luque F,Gómez-Rodríguez M V,Carreras A,Valderrama R,Begara-Morales J C,Hernández L E,Corpas F J,Barroso J B.2011.Functional analysis of superoxide dismutases(SODs)in sunflower under biotic and abiotic stress conditions.Identification of two new genes of mitochondrial Mn-SOD.J Plant Physiol,168(11):1303-1308.
Guo Shao-jian.2016.Analysis of transcription and protein expression of VvHY5 and VvCOP1 in grape peel under light removal condition[Ph.D.Dissertation].Wuhan:Graduate University of Chinese Academy of Sciences.(in Chinese)郭少剑.2016.去光条件下葡萄果皮中VvHY5和VvCOP1的转录和蛋白表达分析[博士论文].武汉:中国科学院研究生院.
Gutteridge S,Gatenby A A.1995.Rubisco synthesis,assembly,mechanism,and regulation.Plant Cell,7(7):809-819.
Huang Yao,Ma Cheng,Wu Nai-hu,Li Zhao-luan.1994.Structural analysis of grape chloroplast rbcL gene.Acta Botanica Sinica,36(6):444-451.(in Chinese)黄瑶,马诚,吴乃虎,李朝銮.1994.葡萄叶绿体rbcL基因的结构分析.植物学报,36(6):444-451.
Jellouli N,Ben Jouira H,Skouri H,Ghorbel A,Gourgouri A,Mliki A.2008.Proteomic analysis of Tunisian grapevine cultivar Razegui under salt stress.J Plant Physiol,165:471-481.
Jin Liang,Chen Shang-wu,Ma Hui-qin.2010.Advances in grape proteomics.China Biotechnology,30(10):100-107.(in Chinese)金良,陈尚武,马会勤.2010.葡萄蛋白质组学研究进展.中国生物工程杂志,30(10):100-107.
Li Feng.2011.Construction and EST analysis of cDNA library from mature peel of Vitis amurensis Rupr[M.D.Dissertation].Harbin:Northeast Forestry University.(in Chinese)李凤.2011.山葡萄成熟果皮cDNA文库的构建及EST分析[硕士论文].哈尔滨:东北林业大学.
Li Ming-liang,Han Yi-fan.2000.Effect of ethylene on the growth and development of plants and inhibition of its biosynthesis by antisense RNA.Seientia Silvae Sinicae,36(4):77-84.(in Chinese)李明亮,韩一凡.2000.乙烯在植物生长发育的抗病反应中的作用及其生物合成的反义抑制.林业科学,36(4):77-84.
Liu G T,Chai F M,Wang Y,Jiang J Z,Duan W,Wang Y T,Wang F F,Li S H,Wang L J.2018.Genome-wide identification and classification of HSF family in grape,and their transcriptional analysis under heat acclimation and heat stress.Horticultural Plant Journal,4(4):133-143.
Mei Yang,Li Hai-lan,Xie Jin,Luo Hong-yi.2007.Ribose-1,5-diphosphate carboxylase/oxygenase(Rubisco).Plant Physiology Communications,(2):363-368.(in Chinese)梅杨,李海蓝,谢晋,罗红艺.2007.核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco).植物生理学通讯,(2):363-368.
Merali S,Boykin C A.2004.S-adenosylmethionine and Pneu-mocystis.FEMS Microbiol Lett,237(1):179-186.
Niu Jiao.2012.Cloning and expression analysis of response genes of powdery mildew of wild Vitis vinifera in China[Ph.D.Dissertation].Yangling:Northwest A&F University.(in Chinese)牛姣.2012.中国野生毛葡萄白粉病响应基因克隆及表达分析[博士论文].杨凌:西北农林科技大学.
Shibli R A,Smith M A L,Kushad M.1997.Headspace ethylene accumulation effects on secondary metabolite production in Vaccinium pahalae cell culture.Plant Growth Reg,23:201-205.
Shimizu T,Tanizawa Y,Mochizuki T,Nagasaki H,Yoshioka T,Toyoda A,Fujiyama A,Kaminuma E,Nakamura Y.2017.Draft Sequencing of the heterozygous diploid genome of satsuma(Citrus unshiu Marc.)Using a Hybrid Assembly Approach Front Genet,8:180.
Spagnolo A,Magnin-Robert M,Alayi T D,Cilindre C,Mercier L,Schaeffer-Reiss C,van Dorsselaer A,Clément C,Fontaine F.2012.Physiological changes in green stems of Vitis vinifera L.cv.Chardonnay in response to esca proper and apoplexy revealed by proteomic and transcriptomic analyses.J Proteome Res,11:461-475.
Wu Jun-lin,Wu Qing-ping,Zhang Ju-mei,Zhang Wen.2015.Progress in L-malic acid biosynthesis.The Food Industry,36(9):225-228.(in Chinese)吴军林,吴清平,张菊梅,张文.2015.L-苹果酸的生物学功能研究新进展.食品工业,36(9):225-228.
Xu Fan.2015.Effect of Brassinolide on sugar unloading during grape maturation[Ph.D.Dissertation].Yangling:Northwest A&F University.(in Chinese)徐凡.2015.油菜素内酯对葡萄成熟过程中糖卸载的影响[博士论文].杨凌:西北农林科技大学.
Yang K A,Moon H J,Kim G T,Ju L C,Hong J C,Oh L C,Yun D J.2003.NDP kinase 2 regulates expression of antioxidant genes in Arabidopsis.Proc Jpn Acad Ser B,79(1):86-91.
Yang Ying-ying,Gao Yang,Zheng Jia-peng,Gan Wu,Xiang Miao-lian,Chen Ming,Chen Jin-yin.2016.Adcances in research on the approach of degradation metabolism of citric acid in citrus fruit and it’s regulation.Acta Agriculturae Universitatis Jiangxinsis,38(3):476-482.(in Chinese)杨滢滢,高阳,郑嘉鹏,甘武,向妙莲,陈明,陈金印.2016.柑橘果实柠檬酸降解代谢途径与调控研究进展.江西农业大学学报,38(3):476-482.
Zhao Jin,Sun Mei-hong,Hu Da-gang,Hao Yu-jin.2015.Molecular cloning and expression analysis of a hexokinase gene MdHXK1 in apple.Acta Horticulturae Sinica,42(8):1437-1447.(in Chinese)赵锦,孙美红,胡大刚,郝玉金.2015.苹果己糖激酶基因MdHXK1的克隆与表达分析.园艺学报,42(8):1437-1447.
Zhang Wen-li,Shen Wen-biao,Ye Mao-bing,Xu Lang-lai.2003.Plant cis and its physiological functions.Plant Physiology Communications,(4):391-398.(in Chinese)张文利,沈文飚,叶茂炳,徐朗莱.2003.植物顺乌头酸酶及其生理功能.植物生理学通讯,(4):391-398.