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作物学报 ›› 2011, Vol. 37 ›› Issue (02): 369-373.doi: 10.3724/SP.J.1006.2011.00369

• 研究简报 • 上一篇    下一篇

圆果种黄麻功能叶总蛋白提取方法及双向电泳体系的优化

陈富成,祁建民*,徐建堂,陈涛,陶爱芬,林培清,陈美霞,郭英,李华丽   

  1. 福建农林大学/ 作物遗传育种与综合利用教育部重点实验室，福建福州350002
  • 收稿日期:2010-07-06 修回日期:2010-10-03 出版日期:2011-02-12 网络出版日期:2010-12-15
  • 基金资助:

    本研究由国家麻类产业技术体系建设(nycytx-19-E05), 国家农业部公益性产业计划(nyhyzx07018-3)和农业部引进先进技术(948计划)项目(2006-G-18-4)资助。

Optimization of Extraction Method and Two-Dimensional Electrophoresis Conditions for Proteome Analysis of Jute Functional Leaf

CHEN Fu-Cheng,QI Jian-Min*,XU Jian-Tang,CHEN Tao,TAO Ai-Fen,LIN Pei-Qing,CHEN Mei-Xia,GUO Ying,LI Hua-Li   

  1. Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China
  • Received:2010-07-06 Revised:2010-10-03 Published:2011-02-12 Published online:2010-12-15

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1602

被引次数

7

摘要： 蛋白质提取方法和电泳条件是蛋白质组学研究中双向电泳的关键。本文初步建立起一套适合圆果种黄麻功能叶总蛋白双向电泳(2-DE)分析的高效提取方法及电泳条件，在双向电泳技术中，比较了TCA丙酮、Tris-base/丙酮、Tris-HCl的蛋白提取方法，并优化了上样量和样品制备方法。结果表明, TCA丙酮法提取蛋白质产量高，达79.0 mg g–1，比Tris-base/丙酮法和Tris-HCl的产量分别高出44.2%和114.1%。该法得到的2-DE图谱背景清晰，蛋白点数最多，约602个，较其他两种方法更适合用于黄麻功能叶总蛋白双向电泳分析；350 mg为最佳上样量。这为黄麻蛋白质组学后续研究奠定了基础。

关键词: 黄麻功能叶, 提取方法, 蛋白质双向电泳(2-DE), 优化上样量

Abstract: The protein extraction methods and electrophoresis conditions are the key facts of two-dimensional electrophoresis in proteomics. In this study, in order to set up a suitable method for jute leaf total protein extraction and the optimized condition for two-dimensional electrophoresis, we compared the protein extraction methods using TCA acetone, Tris-base acetone and Tris-HCl. We also optimized the sample loading amount and sample preparation approach. The results showed that protein productivity reached 79.0 mg g–1 at most by TCA acetone method, and 44.2% and 114.1% higher than by other two methods, respectively. Besides, the 2-DE image background was the clearest and the protein spots were the most reaching about 602 by TCA acetone method, which is much more suitable as extraction method for jute leaf protein two-dimensional electrophoresis. The most suitable sample loading quantity was 350 mg, which lays a foundation for further study of jute proteomics. The construction of two-dimensional electrophoresis system can be use to study the differences of protein expression on normal material and mutant of jute, and promote the application of proteomics in jute mutant.

Key words: Jute leaf, Extraction method, Protein two-dimensional electrophoresis (2-DE), Optimizing of loading amount

[1]O’Farrell P H. High resolution two-dismensional electrophoresis of proteins. Biol Chem, 1975, 250: 4007–4021
[2]Cutler P, Bell D J, Birrell H C, Connelly J C, Connor S C, Holmes E, Mitchell B C, Monté S Y, Neville B A, Pickford R, Polley S, Schneider K, Skehel J M. An integrated proteomic approach to studying glomerular nephrotoxicity. Electrophoresis, 1999, 20: 3647–3658
[3]Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 2000, 21: 1037–1053
[4]Zeng G-J(曾广娟), Li C-M(李春敏), Zhang X-Z(张新忠), Teng Y-L(滕云龙), Dong W-X(董文轩). Efficient protein extraction method from apple leaves for apple proteomic analysis using two-dimensional electrophoresis analysis. Chin J Chromatography (色谱), 2009, 27(4): 484–488(in Chinese with English abstract)
[5]Xu C P, Xu Y, Huang B R. Protein extraction for two-dimensional Gel electrophoresis of proteomic profiling in turfgrass. Crop Sci, 2008, 48: 1608–1614
[6]Gan L(甘露), Li D-R(李殿荣), Zang X(臧新), Fu C-H(付春华), Yu LJ(余龙江), Li M-T(栗茂腾). Construction of protein two-dimensional polyacrylamide Gel electrophoresis system for Brassica napus. Acta Agron Sin (作物学报), 2010, 36(4): 612–619 (in chinese with English abstract)
[7]Huang-Pu H-Y(皇甫海燕), Guan C-Y(官春云), Guo B-S(郭宝顺), Zhang X-Y(张秀英). Progress in proteomics and plant proteomics research. Crop Res (作物研究), 2006, (5): 577–581 (in Chinese with English abstract)
[8]Parker T, Flowers T J, Moore A L, Harpham N V. An accurate and reproducible method for proteome profiling of the effects of salt stress in the rice leaf lamina. J Exp Bot, 2006, 57: 1109–1118
[9]Kim S T, Kim S G, Kang Y H, Wang Y, Kim J Y, Yi N, Kim J K, Rakwal R, Koh H J, Kang K Y. Proteomics analysis of rice lesion mimic mutant (sp/1) reveals tightly localized probenazole induced protein (PBZ1) in cells undergoing programmed cell death. Proteome Res, 2008, 7: 1750–1760
[10]Gallardo K, Job C, Groot S P C, Puype M, Demol H, Vandekerckhove J, Job D. Proteomics of Arabidopsis seed germination: A comparative study of wild-type and gibberellin-deficient seeds. Plant Physiol, 2002, 129: 823–837
[11]Jiang Y Q, Yang B, Harris N S, Deyholos M K. Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. J Exp Bot, 2007, 58: 3591–3607
[12]Amme S, Rutten T, Melzer M, Sonsmann G, Vissers J P, Cschlesier B, Mock H P. A proteome approach defines protective functions of tobacco leaf trichomes. Proteomics, 2005, 5: 2508–2518
[13]Natarajan S, Xu C, Caperna T J, Garrett W M. Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins. Anal Biochem, 2005, 342: 214–220
[14]Xie C J, Wang D, Yang X Y. Protein extraction methods compatible with proteomic analysis for the cotton seedling. Crop Sci, 2009, 49: 395–402
[15]Fountoulakis M. ProteOmics: Current techno1ogies and applications in neurological disorders and toxicology. Amino Acids, 2001, 21: 363–381
[16]Fountou1akis M, Takacs B. Enrichment and proteomic analysis of low-abundance bacterial proteins. Methods Enzymol, 2002, 358: 288–306
[17]Lei J, Lin H, Fountoulakis M. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Chromatogr A, 2004, 1023: 317–320
[18]Carpentier S C, Witters E, Laukens K, Deckers P, Swennen R, Panis B. Preparation of protein extracts from recalcitrant plant tissues: An evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics, 2005, 5: 2497–2507
[19]Gorg A, Weiss W, Dunn M J. Current two-dimensional electrophoresis technology for proteomics. Proteomics, 2004, 4: 3665–3685
[20]Shaw M M, Riederer B M. Sample preparation for twodimensional gel electrophoresis. Proteomics, 2003, 3: 1408–1417
[21]Damerval C, Vienne D D, Zivy M, Thiellement H. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis, 1986, 7: 52–54
[22]Rabilloud T. Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis, 1998, 19: 758–760
[23]Gu R S Liu Q L, Chen X M, Jiang X N. An improved method of 2D electrophoresisfor protein analysis of woody plants. J Beijing For Univ, 1999, 16(2): 171–174
[24]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248–254
[25]Guo J-L(郭晋隆), Ye B-Y(叶冰莹), Huang Q-H(黄庆煌), Chen Y-Q(陈由强), Chen R-K(陈如凯). Improvement of extraction method and two-dimensional electrophor esis conditions for proteome study of sugar cane leaves. Biotechnol Bull (生物技术通报), 2008, 5: 112–116 (in Chinese with English abstract)
[26]Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680–685
[27]Wang J-Y(王经源), Chen S-Y(陈舒奕), Liang Y-Y(梁义元), Lin W-X(林文雄). Improvement of ISO-DALT electrophoresis system. Fujian Agric For Univ (福建农林大学学报), 2006, 35(2): 187–190 (in Chinese with English abstract)
[28]Gao X(高璇), Gao Li-Y(高利艳), Yan Y-M(晏月明). Optimization of extraction method of wheat grain gluteninsand two-dimensional electrophoretic analysis. J Treticeae Crops (麦类作物学报), 2008, 28(6): 971–976 (in Chinese with English abstract)
[29]Jin Y(金艳), Xu H-X(许海霞), Xu Y-Y(徐圆圆), Shang X-L(尚晓丽), Cui D-Q(崔党群). Effect of different extraction protein methods on two-dimensional electrophoresis of wheat leaf protein. J Triticeae Crops (麦类作物学报), 2009, 29(6): 1083–1087 (in Chinese with English abstract)
[30]Vincent D, Wheatley MD, Cramer G R. Optimization of protein extraction and solubilization for mature grape berry clusters. Electrophoresis, 2006, 27: 1853–1865
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