青海高原耐旱蚕豆品种青海13号响应干旱胁迫蛋白质组学分析
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摘要
蛋白质组学研究在功能基因组时代发挥着越来越重要的作用,利用双向电泳技术和质谱鉴定技术,可大量研究作物逆境胁迫后蛋白质组的变化,增加作物响应干旱胁迫机制的认识和理解。为探讨一种抗旱性蚕豆青海13号品种的耐旱机制,本研究对其幼苗期进行干旱胁迫处理,应用上述技术进行差异蛋白质组分析,经t检验发现32个差异表达蛋白点,部分呈现上调表达,部分呈现下调表达,还有7个消失蛋白点和1个新增蛋白点。采用MALDI-TOF/TOF鉴定和生物信息学分析发现,成功鉴定的21个蛋白点按其所参与的代谢途径和生化功能可分为七大类,参与信号转导的2个,参与自由基清除的1个,参与防卫反应的1个,参与代谢的8个,参与蛋白加工的1个,参与光合的5个,未知功能蛋白3个。22 kD干旱诱导蛋白、应激诱导蛋白、17.5 kD一级热激蛋白、超氧化物歧化酶是与抗旱性有直接关联的蛋白点,其相对表达量的上调可能是青海13号蚕豆具有较强抗旱性的重要原因。
Proteomics is playing an increasingly important role in the functional genomics era. Two-dimensional electrophoresis and mass spectrometry can be used to study the changes of proteome of crop under stress and increase the recognization and comprehension of crop response to drought stress. In order to explore the mechanism of drought resistance of Vicia faba L. variety ‘Qinghai 13', we treated the seedlings with three days for water stress and analyzed by two-dimensional gel electrophoresis combined with mass spectrometry analysis. By t-test, 32 differentially expressed proteins spots were detected between normal and drought-stress treatments, respectively, including up-and down-regulated proteins, seven disappear protein spots and a new protein spot. Twenty-one differentially expressed proteins in seven function categories were identified and confirmed by MALDI-TOF/TOF. Among them, two participated in information transfer, one in oxygen radical scavenging, one in protective response, eight in energy metabolism, one in protein processing, five in photosynthesis, and three unknown in function. These results indicate that drought-inducible 22 kD protein, stress-inducible protein, superoxide dismutase and 17.5 kD class HSP are directly related with drought resistance, which may be the important reasons for strong drought resistance of ‘Qinghai 13'.
Proteomics is playing an increasingly important role in the functional genomics era. Two-dimensional electrophoresis and mass spectrometry can be used to study the changes of proteome of crop under stress and increase the recognization and comprehension of crop response to drought stress. In order to explore the mechanism of drought resistance of Vicia faba L. variety ‘Qinghai 13', we treated the seedlings with three days for water stress and analyzed by two-dimensional gel electrophoresis combined with mass spectrometry analysis. By t-test, 32 differentially expressed proteins spots were detected between normal and drought-stress treatments, respectively, including up-and down-regulated proteins, seven disappear protein spots and a new protein spot. Twenty-one differentially expressed proteins in seven function categories were identified and confirmed by MALDI-TOF/TOF. Among them, two participated in information transfer, one in oxygen radical scavenging, one in protective response, eight in energy metabolism, one in protein processing, five in photosynthesis, and three unknown in function. These results indicate that drought-inducible 22 kD protein, stress-inducible protein, superoxide dismutase and 17.5 kD class HSP are directly related with drought resistance, which may be the important reasons for strong drought resistance of ‘Qinghai 13'.
引文
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[11]芮海云,庄凯,沈振国,张芬琴.两个箭舌豌豆品种根响应镉胁迫的蛋白质组学分析.植物生理学报,2016,52:1089-1098.Rui H Y,Zhuang K,Sheng Z G,Zhang F Q.Proteomics analysis of cadmium stress responses in the roots of two Vicia sativa varieties differing in Cd tolerance.Acta Phytophysiol Sin,2016,52:1089-1098(in Chinese with English abstract).
[12]李萍,张雁霞,刘玉皎.干旱胁迫对抗旱性蚕豆幼苗生长特性影响及叶片差异蛋白质组学研究.基因组学与应用生物学,2017,36:340-351.Li P,Zhang Y X,Liu Y J.Effects of drought stress on seeding growth characteristics of drought resistance faba bean(Vicia faba L.)and study on differential proteomics of leaves.J Genom Appl Biol,2017,36:340-351(in Chinese with English abstract).
[13]张小静.马铃薯块茎蛋白质双向电泳技术体系建立及发育相关蛋白质的分析.甘肃农业大学硕士学位论文,甘肃兰州,2008.Zhang X J.Establishment of Two-dimensional Electrophoresis System for Analysis of Proteins during Potato(Solanum tuberosum L.)Tuber Development.MS Thesis of Gansu Agricultural University,Lanzhou,Gansu,China,2008(in Chinese with English abstract).
[14]Demirevska K,Zasheva D,Dimitrov R,Simova-Stoilova L,Stamenova M,Feller U.Drought stress effects on rubisco in wheat:changes in the rubisco large subunit.Acta Physiol Plant,2009,31:1129-1138.
[15]Salekdeh G H,Siopongco J,Wade L J,Ghareyazie B,Benett J.Proteomic analysis of rice leaves during drought stress and recovery.Proteomics,2002,2:1131-1145.
[16]Mohammadi P P,Moieni A,Hiraga S.Organ-specific proleomic analysis of drought-stressed soybean seedling.J Proteomics,2012,75:1960-1923.
[17]Mustafa G,Komatsu S.Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress.Front Plant Sci,2014,5:627.
[18]Ma H Y,Song L R,Huang Z G.Comparative proteomic analysis reveals molecular mechanism of seedling roots of different salt tolerant soybean genotypes in responses to salinity stress.EuPAOpen Proteomics,2014,4:40-57.
[19]Celis J E,Gromov P.2D Protein electrophoresis:can it be perfected?Curr Opin Biotechnol,1999,10:16-21.
[20]Desclos M,Dubousset L,Etienne P,Le Caherec F,Satoh H,Bonnefoy J,Ourry A,Avice J C.A proteomic profiling approach to reveal a novel role of Brassica napus drought 22 kD/watersoluble chlorophyll-binding protein in young leaves during nitrogen remobilization induced by stressful conditons.Plant Physiol,2008,147:1830-1844.
[21]Balestrasse K B,Gardey L,Gallego S M.Response of antioxidant defence systerm in soybean nodules and roots subjected to cadmium stress.Plant Physiol,2001,28:497-453.
[22]Malgorzata G,Waldemar B.Effects of a short-term hypoxic treatment followed by re-aeration on free radicals level and anti-oxidative enzymes in lupine roots.Plant Physiol Biochem,2004,42:233-240.
[23]Hernandez J A,Jimenez A,Mullineaux P,Sevilla F.Tolerance of pea(Pisum sativum L.)to long-term salt stress is associated with induction of antioxidant defences.Plant Cell Environ,2000,23:853-862.
[24]严顺平.水稻响应盐胁迫和低温胁迫的蛋白质组研究.中国科学院博士学位论文,上海,2006.Yan S P.Proteomic Analysis of Salt Stress Responsive and Chilling Stress Response in Rice.PhD Dissertation of Graduate University of the Chinese Academy of Sciences,Shanghai,China,2006(in Chinese with English abstract).
[25]Hajheidari M,Abdollahian-Noghabi M,Askari H,Heidari M,Sadeghian S Y,Ober E S,Salekdeh G H.Proteome analysis of sugar beet leaves under drought stress.Proteomics,2005,5:950-960.
[26]Wang W,Vinocur B,Shoseyov O,Altman A.Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response.Trends Plant Sci,2004,9:244-252.
[27]Sato Y,Yokoya S.Enhanced to tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7.Plant Cell Rep,2008,27:329-334.
[28]丁伟.水稻干旱胁迫蛋白质组相关数据和生物信息分析研究.华中农业大学硕士学位论文,湖北武汉,2009.Ding W.Analysis of Bioinformation and Data Related to Proteome of Rice under Drought Stress.MS Thesis of Huazhong Agricultural University,Wuhan,Hubei,China,2009(in Chinese with English abstract).
[29]舒烈波.水稻叶片响应干旱和渗透胁迫的蛋白质组学研究.华中农业大学博士学位论文,湖北武汉,2010.Shu L B.Proteomic Analysis of Rice Leaves in Response to Drought and Osmotic Stress.PhD Dissertation of Huazhong Agricultural University,Wuhan,Hubei,China,2010(in Chinese with English abstract).
[30]李丽芳,罗晓芳,王华芳.植物抗旱基因工程研究进展.西北林学院学报,2004,19:53-57.Li L F,Luo X F,Wang H F.Advances in the studies of gene engineering on plant drought-resistance.J Northwest For Univ,2004,19:53-57(in Chinese with English abstract).
[31]Weaver L M,Herrmann K M.Dynamics of the shikimate pathway in plants.Trends Plant Sci,1997,2:346-351.
[32]Diaz J,Bernal A,Pomar F,Merino F.Induction of shikimate dehydrogenase and peroxidase in pepper(Capsicum annuum L.)seedlings in response to copper stress and its relation to lignification.Plant Sci,2001,161:179-188.
[33]Rospert S,Dubaquie Y,Gautschi M.Nascentpolypeptideassociated complex.Cell Mol Life Sci,2002,59:1632-1639.
[34]Chaves M M,Flexas J,Pinheiro C.Photosynthesis under drought and salt stress:Regulation mechanisms from whole plant to cell.Ann Bot,2009,103:551-560.
[35]Reddy A R,Chaitanya K V,Vivekanandan M.Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants.J Plant Physiol,2004,161:1189-1202.
[36]Thomashow M F.Plant cold acclimation:freezing tolerance genes and regulatory mechanisms.Annu Rev Plant Physoil Plant Mol Biol,1999,50:571-599.
[37]Li X J,Yang M F,Chen H,Qu L Q,Chen F,Shen S H.Abscisic acid pretreatment enhances salt tolerance of rice seedlings:proteomic evidence.Biochim Biophys Acta,2010,1804:929-940.
[2]Marie P R,Vartavian N,Sallantin M.Characterization of a novel protein induced by progressive or rapid drought and salinity in Brassica napus leaves.Plant Physiol,1992,100:1486-1493.
[3]Ingram J,Bartels D.The molecular basis of dehydration tolerance in plant.Annu Rev Plant Physoil Plant Mol Biol,1996,47:377-403.
[4]Levenfors J P,Wikstr M M,Persson L,Gerhardson B.Pathogenicity of aphanomyces spp from different leguminous crops in Sweden.Eur J Plant Pathol,2003,10:535-543.
[5]Agrawal G K,Pedreschi R,Barkla B J,Bindschedler L V,Cramer R,Sarkar A,Renaut J,Job D,Rakwal R.Translational plant proteomics:A perspective.J Proteomics,2012,75:4588-4601.
[6]DO Thanh-Trung,李健,张风娟,杨丽涛,李杨瑞,邢永秀.甘蔗与抗旱性相关差异蛋白质组分析.作物学报,2017,43:1337-1346.DO T T,Li J,Zhang F J,Yang L T,Li Y R,Xing Y X.Analysis of differential proteome in relation to drought resistance in Sugarcane.Acta Agron Sin,2017,43:1337-1346(in Chinese with English abstract).
[7]Mohammadi P P,Moieni A,Hiraga S,Komatsu S.Organ-specific proteomic analysis of drought-stressed soybean seedlings.J Proteomics,2012,75:1906-1923.
[8]Zadra?nik T,Hollung K,Egge-Jacobsen W.Differential proteomic analysis of drought stress response in leaves of common bean(Phaseolus vulgaris L.).J Proteomics,2013,78:254-272.
[9]Badowiec A,Weidner S.Proteomic changes in the roots of germinating Phaseolus vulgaris seeds in response to chilling stress and post-stress recovery.J Plant Physiol,2014,2:389-398.
[10]熊军波,杨青川,蔡化,田宏,张贺山,刘洋.紫花苜蓿根响应盐胁迫的比较蛋白质组学分析.湖北农业科学,2015,54:5422-5428.Xiong J B,Yang Q H,Cai H,Tian H,Zhang H S,Li Y.Comparative proteomic analysis of salt-stress response of alfalfa proteins in Root.J Hubei Agric Sci,2015,54:5422-5428(in Chinese with English abstract).
[11]芮海云,庄凯,沈振国,张芬琴.两个箭舌豌豆品种根响应镉胁迫的蛋白质组学分析.植物生理学报,2016,52:1089-1098.Rui H Y,Zhuang K,Sheng Z G,Zhang F Q.Proteomics analysis of cadmium stress responses in the roots of two Vicia sativa varieties differing in Cd tolerance.Acta Phytophysiol Sin,2016,52:1089-1098(in Chinese with English abstract).
[12]李萍,张雁霞,刘玉皎.干旱胁迫对抗旱性蚕豆幼苗生长特性影响及叶片差异蛋白质组学研究.基因组学与应用生物学,2017,36:340-351.Li P,Zhang Y X,Liu Y J.Effects of drought stress on seeding growth characteristics of drought resistance faba bean(Vicia faba L.)and study on differential proteomics of leaves.J Genom Appl Biol,2017,36:340-351(in Chinese with English abstract).
[13]张小静.马铃薯块茎蛋白质双向电泳技术体系建立及发育相关蛋白质的分析.甘肃农业大学硕士学位论文,甘肃兰州,2008.Zhang X J.Establishment of Two-dimensional Electrophoresis System for Analysis of Proteins during Potato(Solanum tuberosum L.)Tuber Development.MS Thesis of Gansu Agricultural University,Lanzhou,Gansu,China,2008(in Chinese with English abstract).
[14]Demirevska K,Zasheva D,Dimitrov R,Simova-Stoilova L,Stamenova M,Feller U.Drought stress effects on rubisco in wheat:changes in the rubisco large subunit.Acta Physiol Plant,2009,31:1129-1138.
[15]Salekdeh G H,Siopongco J,Wade L J,Ghareyazie B,Benett J.Proteomic analysis of rice leaves during drought stress and recovery.Proteomics,2002,2:1131-1145.
[16]Mohammadi P P,Moieni A,Hiraga S.Organ-specific proleomic analysis of drought-stressed soybean seedling.J Proteomics,2012,75:1960-1923.
[17]Mustafa G,Komatsu S.Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress.Front Plant Sci,2014,5:627.
[18]Ma H Y,Song L R,Huang Z G.Comparative proteomic analysis reveals molecular mechanism of seedling roots of different salt tolerant soybean genotypes in responses to salinity stress.EuPAOpen Proteomics,2014,4:40-57.
[19]Celis J E,Gromov P.2D Protein electrophoresis:can it be perfected?Curr Opin Biotechnol,1999,10:16-21.
[20]Desclos M,Dubousset L,Etienne P,Le Caherec F,Satoh H,Bonnefoy J,Ourry A,Avice J C.A proteomic profiling approach to reveal a novel role of Brassica napus drought 22 kD/watersoluble chlorophyll-binding protein in young leaves during nitrogen remobilization induced by stressful conditons.Plant Physiol,2008,147:1830-1844.
[21]Balestrasse K B,Gardey L,Gallego S M.Response of antioxidant defence systerm in soybean nodules and roots subjected to cadmium stress.Plant Physiol,2001,28:497-453.
[22]Malgorzata G,Waldemar B.Effects of a short-term hypoxic treatment followed by re-aeration on free radicals level and anti-oxidative enzymes in lupine roots.Plant Physiol Biochem,2004,42:233-240.
[23]Hernandez J A,Jimenez A,Mullineaux P,Sevilla F.Tolerance of pea(Pisum sativum L.)to long-term salt stress is associated with induction of antioxidant defences.Plant Cell Environ,2000,23:853-862.
[24]严顺平.水稻响应盐胁迫和低温胁迫的蛋白质组研究.中国科学院博士学位论文,上海,2006.Yan S P.Proteomic Analysis of Salt Stress Responsive and Chilling Stress Response in Rice.PhD Dissertation of Graduate University of the Chinese Academy of Sciences,Shanghai,China,2006(in Chinese with English abstract).
[25]Hajheidari M,Abdollahian-Noghabi M,Askari H,Heidari M,Sadeghian S Y,Ober E S,Salekdeh G H.Proteome analysis of sugar beet leaves under drought stress.Proteomics,2005,5:950-960.
[26]Wang W,Vinocur B,Shoseyov O,Altman A.Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response.Trends Plant Sci,2004,9:244-252.
[27]Sato Y,Yokoya S.Enhanced to tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7.Plant Cell Rep,2008,27:329-334.
[28]丁伟.水稻干旱胁迫蛋白质组相关数据和生物信息分析研究.华中农业大学硕士学位论文,湖北武汉,2009.Ding W.Analysis of Bioinformation and Data Related to Proteome of Rice under Drought Stress.MS Thesis of Huazhong Agricultural University,Wuhan,Hubei,China,2009(in Chinese with English abstract).
[29]舒烈波.水稻叶片响应干旱和渗透胁迫的蛋白质组学研究.华中农业大学博士学位论文,湖北武汉,2010.Shu L B.Proteomic Analysis of Rice Leaves in Response to Drought and Osmotic Stress.PhD Dissertation of Huazhong Agricultural University,Wuhan,Hubei,China,2010(in Chinese with English abstract).
[30]李丽芳,罗晓芳,王华芳.植物抗旱基因工程研究进展.西北林学院学报,2004,19:53-57.Li L F,Luo X F,Wang H F.Advances in the studies of gene engineering on plant drought-resistance.J Northwest For Univ,2004,19:53-57(in Chinese with English abstract).
[31]Weaver L M,Herrmann K M.Dynamics of the shikimate pathway in plants.Trends Plant Sci,1997,2:346-351.
[32]Diaz J,Bernal A,Pomar F,Merino F.Induction of shikimate dehydrogenase and peroxidase in pepper(Capsicum annuum L.)seedlings in response to copper stress and its relation to lignification.Plant Sci,2001,161:179-188.
[33]Rospert S,Dubaquie Y,Gautschi M.Nascentpolypeptideassociated complex.Cell Mol Life Sci,2002,59:1632-1639.
[34]Chaves M M,Flexas J,Pinheiro C.Photosynthesis under drought and salt stress:Regulation mechanisms from whole plant to cell.Ann Bot,2009,103:551-560.
[35]Reddy A R,Chaitanya K V,Vivekanandan M.Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants.J Plant Physiol,2004,161:1189-1202.
[36]Thomashow M F.Plant cold acclimation:freezing tolerance genes and regulatory mechanisms.Annu Rev Plant Physoil Plant Mol Biol,1999,50:571-599.
[37]Li X J,Yang M F,Chen H,Qu L Q,Chen F,Shen S H.Abscisic acid pretreatment enhances salt tolerance of rice seedlings:proteomic evidence.Biochim Biophys Acta,2010,1804:929-940.