大豆叶片蛋白响应干旱胁迫的双向电泳分析
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摘要
大豆是我国重要的农作物之一,蛋白质组学技术是解析植物响应逆境胁迫的重要工具之一。为进一步推进对响应干旱胁迫的大豆叶片蛋白的鉴定工作,以大豆幼苗的叶片为研究对象,用甘露醇溶液模拟干旱胁迫,处理24 h后提取叶片蛋白粉进行双向电泳分离和图谱差异分析。结果表明:采取三氯醋酸(TCA)-丙酮沉淀法提取的叶片蛋白进行双向电泳分离能够得到稳定清晰的双向电泳蛋白图谱。采取PD-Quest软件对干旱胁迫下的大豆叶片蛋白表达图谱进行差异分析,共检测和匹配425个大豆叶片蛋白点,其中差异表达的蛋白点有101个,60个表达量上调,41个表达量下调。本研究结果表明有一部分大豆叶片蛋白响应了干旱胁迫。
Soybean is one of the important crops in China. Proteomics technology is one of the important tools to analyze plantresponse to stress. To lay the foundation for further identification of homologous proteins of soybean leaf protein in response todrought stress,the leaves of soybean seedlings were used as research objects. The mannitol solution was used to simulatedrought stress. After 24 h treatment,the leaf protein powder was extracted for two-dimensional electrophoresis separation andspectral difference analysis. The results showed that the leaf protein was extracted by trichloroacetic acid-acetone precipitationmethod for two-dimensional electrophoresis analysis to obtain a stable and clear two-dimensional electrophoresis protein map.PD-Quest software was used to analyze the protein expression profiles of soybean leaves under drought stress. A total of 425 soybean leaf protein spots were detected and matched. There were 101 differentially expressed protein spots,among which 60 were up-regulated,and 41 were down-regulated. The results of this study indicated that there were some soybean leaf proteinsresponded to drought stress.
Soybean is one of the important crops in China. Proteomics technology is one of the important tools to analyze plantresponse to stress. To lay the foundation for further identification of homologous proteins of soybean leaf protein in response todrought stress,the leaves of soybean seedlings were used as research objects. The mannitol solution was used to simulatedrought stress. After 24 h treatment,the leaf protein powder was extracted for two-dimensional electrophoresis separation andspectral difference analysis. The results showed that the leaf protein was extracted by trichloroacetic acid-acetone precipitationmethod for two-dimensional electrophoresis analysis to obtain a stable and clear two-dimensional electrophoresis protein map.PD-Quest software was used to analyze the protein expression profiles of soybean leaves under drought stress. A total of 425 soybean leaf protein spots were detected and matched. There were 101 differentially expressed protein spots,among which 60 were up-regulated,and 41 were down-regulated. The results of this study indicated that there were some soybean leaf proteinsresponded to drought stress.
引文
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[4]董兴月,林浩,刘丽君,等.干旱胁迫对大豆生理指标的影响[J].大豆科学,2011,30(1):83-88.(Dong X Y,Lin H,Liu LJ,et al. Effects of drought stress on physiological indexes of soy-bean[J]. Soybean Science,2011,30(1):83-88.)
[5]王燕平,王晓梅,侯国强,等.干旱胁迫对不同生态型大豆生理生化特征的影响[J].中国农学通报,2014,30(12):93-100.(Wang Y P,Wang X M,Hou G Q,et al. Effects of drought stresson physiological and biochemical characteristics of different eco-types of soybean[J]. Chinese Agricultural Science Bulletin,2014,30(12):93-100.)
[6]王春艳,庞艳梅,李茂松,等.干旱胁迫对大豆气孔特征和光合参数的影响[J].中国农业科技导报,2013,15(1):109-115.(Wang C Y,Pang Y M,Li M S,et al. Effects of drought stress onstomatal characteristics and photosynthetic parameters of soybean[J]. China Agricultural Science and Technology Review,2013,15(1):109-115.)
[7] Mohammadi P P,Moieni A,Hiraga S,et al. Organ-specific pro-teomic analysis of drought-stressed soybean seedlings[J]. Journalof Proteomics,2012,75:1906-1923.
[8] Khatoon A,Rehman S,Hiraga S,et al. Organ-specific proteomics a-nalysis for identification of response mechanism in soybean seed-lings under flooding stress[J]. Journal of Proteomics,2012,75:5706-5723.
[9] Komatsu S,Yamamoto A,Nakamura T,et al. Comprehensive a-nalysis of mitochondria in roots and hypocotyls of soybean underflooding stress using proteomics and metabolomics techniques[J].Journal of Proteome Research,2011,10(9):3993-4004.
[10] Ma H Y,Song L R,Shu Y J,et al. Comparative proteomic analysisof seedling leaves of different salt tolerant soybean genotypes[J].Journal of Proteomics,2012,75:1529-1546.
[11]王文军.大豆种子冷害和PEG引发的蛋白质组学分析[D].呼和浩特:内蒙古农业大学,2008.(Wang W J. Proteomic anal-ysis of soybean seed chilling injury and PEG induced[D]. Ho-hhot:Inner Mongolia Agricultural University,2008.)
[12] Damerval C,de Vienne D,Zivy M. Technical improvements in two-dimensional electrophoresis increase the level of genetic variationdetected in wheat seeding proteins[J]. Electrophories,1986,7(1):52-54.
[13] Braford M M. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.
[14] Park I,Ndjomou J,Wen Y,et al. Inhibition of HCV replicationby oxysterol-binding protein-related protein 4(ORP4)through in-teraction with HCV NS5B and Alteration of lipid droplet formation[J]. PLo S One,2013,8(9):e75648.
[15] Herman E M,Helm R M,Jung R,et al. Genetic modification re-moves an immune dominant allergen from soybean[J]. Plant Physi-ology,2003,132(1):36-43.
[16] Hurkman W J,Tanaka C K. Solubilization of plant membrane pro-teins for analysis by two-dimensional gel electrophoresis[J]. PlantPhysiology,1986,81(3):802-806.
[17] Meyer Y,Grosset J,Chartier Y,et al. Preparation by two-dimen-sional electrophoresis of proteins for antibody production:Antibod-ies against proteins whose synthesisis reduced by auxin in tobaccomesophyll protoplasts[J]. Electrophoresis, 1988,9(11):704-712.
[18] Wang W,Vignani R,Scali M,et al. Removal of lipid contaminantsby organic solvents from oilseed protein extract prior to electropho-resis[J]. Analytical Biochemistry,2004,329(1):139-141.
[19] Hurkman W J,Tanaka C K. Solubilization of plant membrane pro-teins for analysis by two-dimensional gel electrophoresis[J]. PlantPhysiology,1986,81(3):802-806.