An Improved System of Two-dimensional Electrophoresis for the Tung Tree Seed Protein Separation

ZHAN Zhi-yong; CHEN Yi-cun; HAN Xiao-jiao; CUI Qin-qin; WANG Yang-dong

Volume 25 Issue 6

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An Improved System of Two-dimensional Electrophoresis for the Tung Tree Seed Protein Separation

1.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China

Received Date: 2012-06-26

Abstract

In order to construct a basis for the tung trees protein research, some key factors which could impact on the results of the protein separation were studied and an improved system of two-dimensional electrophoresis were built successfully including the suitable protein extraction method (TCA-acetone combined with phenol), the solution buffer (7 mol·L-1 urea, 2 mol·L-1 thiourea, 4% CHAPS, 65 mmol·L-1 DTT and 2% IPG buffer), the volumes of loading protein (700 μg) in IEF and the better conditions for the SDS-PAGE (15% separation gels and constant 5W for 8 h).
- tung trees,
- two-dimensional electrophoresis (2-DE),
- proteomics

References

[1]	蔡金标,丁建祖,陈必勇. 中国油桐品种、类型的分类[J]. 经济林研究,1997, 15(4): 47-50
[2]	凌麓山,何方,方嘉兴,等. 中国油桐品种分类的研究[J]. 经济林研究,1991, 9(2): 1-8
[3]	刘翠峰,王彦英,翟运吾,等. 油桐豫桐1号等3个优良家系的选育[J]. 经济林研究,1996, 14(1): 45-47
[4]	吕平会,李龙山,谢复明,等. 油桐优良单株选择[J]. 经济林研究,1993, 11(2): 88-91
[5]	龚榜初,蔡金标. 油桐育种研究的进展[J]. 经济林研究,1996, 14(1): 51-53
[6]	刘益兴,贺赐平,李正茂,等. 不同繁殖方式对油桐树体结构及产量的影响[J]. 中南林业科技大学学报,2010, 30(5): 61-66
[7]	隆振雄. 油桐北移引种幼苗越冬相关性状的遗传分析[J]. 西北林学院学报,1996, 11(3): 31-35
[8]	涂炳坤,郭刚奇,徐正红,等. 油桐数量性状的主成分分析及分类[J]. 华中农业大学学报,1994, 13(3): 296-300
[9]	夏逍鸿,卢龙高. 油桐主要经济性状与产油相关分析[J]. 浙江林业科技,1992, 12(1): 32-33
[10]	吴开云,费学谦,姚小华. 油桐DNA快速提取以及RAPD扩增的初步研究[J]. 经济林研究,1998, 16(3): 28-30
[11]	汪阳东,李元,李鹏. 油桐桐酸合成酶基因克隆和植物表达载体构建[J]. 浙江林业科技,2007, 27(2): 1-5
[12]	李鹏,汪阳东,陈益存,等. 油桐ISSR-PCR最佳反应体系的建立[J]. 林业科学研究,2008, 21(2): 194-199
[13]	李建安,孙颖,陈鸿鹏,等. 油桐LFAFY同源基因片段的克隆与分析[J]. 中南林业科技大学学报,2008, 28(4): 21-26
[14]	龙红旭,谭晓风,陈洪,等. 油桐油体蛋白基因的克隆及序列分析[J]. 中南林业科技大学学报,2010, 30(4): 31-38
[15]	Bradford M M. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of proteindye binding[J]. Analytical Biochemistry, 1976, 72(1-2): 248-254
[16]	Magni C, Scarafoni A, Herndl A, et al. Combined 2D electrophoretic approaches for the study of white lupin mature seed storage proteome [J]. Phytochemistry, 2007, 68(7): 997-1007
[17]	Wang X Q, Yang P F, Zhang X F, et al. Proteomic analysis of the cold stress response in the moss, Physcomitrella patens[J]. Proteomics, 2009, 9(19): 4529-4538
[18]	Han F, Chen H, Li X J, et al. A comparative proteomic analysis of rice seedlings under various high-temperature stress [J]. Biochimica et Biophysica Acta, 2009, 1794(11): 1625-1634
[19]	Liu H, Liu Y J, Yang M F, et al. A comparative analysis of embryo and endosperm proteome from seeds of Jatropha curcas[J]. Journal of Integrative Plant Biology, 2009, 51(9): 850-857
[20]	Blackstock W P, Weir M P. Proteomics: quantitative and physical mapping of cellular proteins [J]. Trends Biotechnol, 1999, 17(3): 121-127
[21]	Rabilloud T. Solubilization of proteins in 2-D electrophoresis: an outline [M]// Link A J. Methods molecular biology, Vol. 112: 2-D proteome analysis protocols.Totowa,NJ:Humana Press Inc.,1999: 9-19
[22]	Saravanan R S, Rose J K. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues [J]. Proteomics, 2004, 4(9): 22-32
[23]	Giavalisco P, Nordhoff E, Lehrach H, et al. Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis [J]. Electrophoresis, 2003, 24(1-2): 207-216
[24]	Hurkman W J, Tanaka C K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis [J]. Plant Physiol, 1986, 81(3): 802-806
[25]	李德军,邓治,陈春柳,等. 植物组织双向电泳样品制备方法研究进展[J]. 中国农学通报, 2009, 25(24): 78-82
[26]	Liu H, Yang Z L, Yang M F, et al. The differential proteome of endosperm and embryo from mature seed of Jatropha curcas[J]. Plant Science, 2011, 181(6): 660-666
[27]	Wang W, Vignani R, Scali M, et al. A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis [J]. Electrophoresis, 2006, 27(13): 2782-2786
[28]	Carpentier S C, Witters E, Laukens K, et al. Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two dimensional gel electrophoresis analysis [J]. Proteomics, 2005, 5(10): 2497-2507
[29]	Shaw M M, Riederer B M. Sample preparation for two-dimensional gel electrophoresis [J]. Proteomics, 2003, 3(8): 1408-1417
[30]	李红兵,康振生. 小麦叶片蛋白质组分析中高丰度蛋白Rubisco酶的快速去除方法研究[J]. 西北农林科技大学学报:自然科学版, 2011, 39(6): 223-228
[31]	Yao Y, Yang Y W, Liu J Y. An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE [J]. Electrophoresis, 2006, 27(22): 1-11
[32]	曹景林,朱龙付,谭家福,等. 适用于蛋白质双向电泳的棉花胚性培养物蛋白质提取技术[J]. 棉花学报,2009, 21(1): 3-9
[33]	王清,产祝龙,秦国政,等. 果实蛋白质组学研究的实验方法[J]. 植物学报, 2009, 44(1): 107-116
[34]	Meyer T S, Lamberts B L. Use of Coomassie Brilliant Blue R250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide-gel strips [J]. Biochim Biophys Acta, 1965, 107(14): 144-145
[35]	Candiano G, Bruschi M, Musante L, et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis [J]. Electrophoresis, 2004, 25(9): 1327-1333
[36]	Rabilloud T, Vuillard L, Gilly C, et al. Silver staining of proteins in polyacrylamide gels: a general overview [J]. Cell Mol Bio, 1990, 40(1): 57-75

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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An Improved System of Two-dimensional Electrophoresis for the Tung Tree Seed Protein Separation

1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China

Received Date: 2012-06-26

Available Online: 2012-12-13

Abstract: In order to construct a basis for the tung trees protein research, some key factors which could impact on the results of the protein separation were studied and an improved system of two-dimensional electrophoresis were built successfully including the suitable protein extraction method (TCA-acetone combined with phenol), the solution buffer (7 mol·L-1 urea, 2 mol·L-1 thiourea, 4% CHAPS, 65 mmol·L-1 DTT and 2% IPG buffer), the volumes of loading protein (700 μg) in IEF and the better conditions for the SDS-PAGE (15% separation gels and constant 5W for 8 h).

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Reference (36)

[1]	蔡金标,丁建祖,陈必勇. 中国油桐品种、类型的分类[J]. 经济林研究,1997, 15(4): 47-50
[2]	凌麓山,何方,方嘉兴,等. 中国油桐品种分类的研究[J]. 经济林研究,1991, 9(2): 1-8
[3]	刘翠峰,王彦英,翟运吾,等. 油桐豫桐1号等3个优良家系的选育[J]. 经济林研究,1996, 14(1): 45-47
[4]	吕平会,李龙山,谢复明,等. 油桐优良单株选择[J]. 经济林研究,1993, 11(2): 88-91
[5]	龚榜初,蔡金标. 油桐育种研究的进展[J]. 经济林研究,1996, 14(1): 51-53
[6]	刘益兴,贺赐平,李正茂,等. 不同繁殖方式对油桐树体结构及产量的影响[J]. 中南林业科技大学学报,2010, 30(5): 61-66
[7]	隆振雄. 油桐北移引种幼苗越冬相关性状的遗传分析[J]. 西北林学院学报,1996, 11(3): 31-35
[8]	涂炳坤,郭刚奇,徐正红,等. 油桐数量性状的主成分分析及分类[J]. 华中农业大学学报,1994, 13(3): 296-300
[9]	夏逍鸿,卢龙高. 油桐主要经济性状与产油相关分析[J]. 浙江林业科技,1992, 12(1): 32-33
[10]	吴开云,费学谦,姚小华. 油桐DNA快速提取以及RAPD扩增的初步研究[J]. 经济林研究,1998, 16(3): 28-30
[11]	汪阳东,李元,李鹏. 油桐桐酸合成酶基因克隆和植物表达载体构建[J]. 浙江林业科技,2007, 27(2): 1-5
[12]	李鹏,汪阳东,陈益存,等. 油桐ISSR-PCR最佳反应体系的建立[J]. 林业科学研究,2008, 21(2): 194-199
[13]	李建安,孙颖,陈鸿鹏,等. 油桐LFAFY同源基因片段的克隆与分析[J]. 中南林业科技大学学报,2008, 28(4): 21-26
[14]	龙红旭,谭晓风,陈洪,等. 油桐油体蛋白基因的克隆及序列分析[J]. 中南林业科技大学学报,2010, 30(4): 31-38
[15]	Bradford M M. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of proteindye binding[J]. Analytical Biochemistry, 1976, 72(1-2): 248-254
[16]	Magni C, Scarafoni A, Herndl A, et al. Combined 2D electrophoretic approaches for the study of white lupin mature seed storage proteome [J]. Phytochemistry, 2007, 68(7): 997-1007
[17]	Wang X Q, Yang P F, Zhang X F, et al. Proteomic analysis of the cold stress response in the moss, Physcomitrella patens[J]. Proteomics, 2009, 9(19): 4529-4538
[18]	Han F, Chen H, Li X J, et al. A comparative proteomic analysis of rice seedlings under various high-temperature stress [J]. Biochimica et Biophysica Acta, 2009, 1794(11): 1625-1634
[19]	Liu H, Liu Y J, Yang M F, et al. A comparative analysis of embryo and endosperm proteome from seeds of Jatropha curcas[J]. Journal of Integrative Plant Biology, 2009, 51(9): 850-857
[20]	Blackstock W P, Weir M P. Proteomics: quantitative and physical mapping of cellular proteins [J]. Trends Biotechnol, 1999, 17(3): 121-127
[21]	Rabilloud T. Solubilization of proteins in 2-D electrophoresis: an outline [M]// Link A J. Methods molecular biology, Vol. 112: 2-D proteome analysis protocols.Totowa,NJ:Humana Press Inc.,1999: 9-19
[22]	Saravanan R S, Rose J K. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues [J]. Proteomics, 2004, 4(9): 22-32
[23]	Giavalisco P, Nordhoff E, Lehrach H, et al. Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis [J]. Electrophoresis, 2003, 24(1-2): 207-216
[24]	Hurkman W J, Tanaka C K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis [J]. Plant Physiol, 1986, 81(3): 802-806
[25]	李德军,邓治,陈春柳,等. 植物组织双向电泳样品制备方法研究进展[J]. 中国农学通报, 2009, 25(24): 78-82
[26]	Liu H, Yang Z L, Yang M F, et al. The differential proteome of endosperm and embryo from mature seed of Jatropha curcas[J]. Plant Science, 2011, 181(6): 660-666
[27]	Wang W, Vignani R, Scali M, et al. A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis [J]. Electrophoresis, 2006, 27(13): 2782-2786
[28]	Carpentier S C, Witters E, Laukens K, et al. Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two dimensional gel electrophoresis analysis [J]. Proteomics, 2005, 5(10): 2497-2507
[29]	Shaw M M, Riederer B M. Sample preparation for two-dimensional gel electrophoresis [J]. Proteomics, 2003, 3(8): 1408-1417
[30]	李红兵,康振生. 小麦叶片蛋白质组分析中高丰度蛋白Rubisco酶的快速去除方法研究[J]. 西北农林科技大学学报:自然科学版, 2011, 39(6): 223-228
[31]	Yao Y, Yang Y W, Liu J Y. An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE [J]. Electrophoresis, 2006, 27(22): 1-11
[32]	曹景林,朱龙付,谭家福,等. 适用于蛋白质双向电泳的棉花胚性培养物蛋白质提取技术[J]. 棉花学报,2009, 21(1): 3-9
[33]	王清,产祝龙,秦国政,等. 果实蛋白质组学研究的实验方法[J]. 植物学报, 2009, 44(1): 107-116
[34]	Meyer T S, Lamberts B L. Use of Coomassie Brilliant Blue R250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide-gel strips [J]. Biochim Biophys Acta, 1965, 107(14): 144-145
[35]	Candiano G, Bruschi M, Musante L, et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis [J]. Electrophoresis, 2004, 25(9): 1327-1333
[36]	Rabilloud T, Vuillard L, Gilly C, et al. Silver staining of proteins in polyacrylamide gels: a general overview [J]. Cell Mol Bio, 1990, 40(1): 57-75

An Improved System of Two-dimensional Electrophoresis for the Tung Tree Seed Protein Separation

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An Improved System of Two-dimensional Electrophoresis for the Tung Tree Seed Protein Separation

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