一种有效检测水稻叶鞘低丰度蛋白的方法

草业学报 ›› 2011, Vol. 20 ›› Issue (3): 192-197.

一种有效检测水稻叶鞘低丰度蛋白的方法

王莹^1,2,崔为同^1,2,杨明峰^1,2,沈世华^1,2*

1.中国科学院北京植物所,北京100093;
2.中国科学院研究生院,北京100049

收稿日期:2010-04-11 出版日期:2011-03-25 发布日期:2011-06-20
通讯作者: E-mail:shshen@ibcas.ac.cn
作者简介:王莹(1985-),女,山东菏泽人,在读硕士。E-mail:wangying8558@hotmail.com
基金资助:
国家高技术研究发展计划项目(No. 2007AA10Z109)资助。

An approach to detect the low-abundant proteins in rice leaf sheath

WANG Ying^1,2, CUI Wei-tong^1,2, YANG Ming-feng^1,2, SHEN Shi-hua^1,2

1.Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China;
2.Graduate University of Chinese Academy of Sciences, Beijing 100049, China

Received:2010-04-11 Online:2011-03-25 Published:2011-06-20

摘要/Abstract

摘要： 蛋白质组学研究中,低丰度蛋白的富集、检测和鉴定是主要的技术难题。植物组织中核酮糖-1,5-二磷酸羧化/加氧酶(rubisco)等高丰度蛋白占细胞全蛋白很大比例,严重影响双向凝胶电泳(2-DE)对低丰度蛋白的检测。为探索一种适用于叶鞘低丰度蛋白的有效检测方法,本研究采用聚乙二醇(PEG)沉淀法以减少rubisco等高丰度蛋白,富集低丰度蛋白。以Mg/NP-40法为对照,分别使用15%和20%PEG分离水稻叶鞘可溶性全蛋白,并将对照和各PEG分离组分进行2-DE。2-DE图谱经软件分析结果表明,20%PEG沉淀法适用于灌浆期叶鞘低丰度蛋白检测。

Abstract: Enrichment of low-abundant proteins and their detection still remain great challenges in proteomics research. The existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in plants, leads to this dilemma. The high-abundant proteins engage a large proportion of the whole-cell proteins and thus make the low-abundant proteins poorly detectable by 2-DE. In this report, we used a protocol for the preparation of whole-cell proteins through polyethylene glycol (PEG) precipitation, to develop an approach of identifying low-abundant proteins in rice leaf sheathes. In a comparison of the 2-DE analyses of protein samples prepared using the Mg/NP-40 method without PEG fractionation, with the 15% and 20% PEG fractionation protocol, a relatively high reproducibility was achieved using the 20% PEG fractionation protocol in terms of protein species and low-abundant proteins.

中图分类号:

王莹,崔为同,杨明峰,沈世华. 一种有效检测水稻叶鞘低丰度蛋白的方法[J]. 草业学报, 2011, 20(3): 192-197.

WANG Ying, CUI Wei-tong, YANG Ming-feng, SHEN Shi-hua. An approach to detect the low-abundant proteins in rice leaf sheath[J]. Acta Prataculturae Sinica, 2011, 20(3): 192-197.

参考文献

[1] 曾嵘, 夏其昌. 蛋白质组学研究进展与趋势[J]. 中国科学院院刊, 2002, 17(3): 166-169.
[2] Phizicky E, Bastiaens, Zhu H, et al. Protein analysis on a proteomic scale[J]. Nature, 2003, 422: 208-215.
[3] Imin N, Kerim T, Rolfe B G, et al. Effect of early cold stress on the maturation of rice anthers[J]. Proteomics, 2004, 4: 1873-1882.
[4] Imin N, Kerim T, Weinman J J, et al. Low temperature treatment at the young microspore stage induces protein changes in rice anthers[J]. Molecular Cellular Proteomics, 2006, 5: 274-292.
[5] Yan S P, Zhang Q Y, Tang Z C, et al. Comparative proteomic analysis provides new insights into chilling stress responses in rice[J]. Molecular Cellular Proteomics, 2006, 5: 484-496.
[6] Adam G C, Sorensen E J, Cravatt B F. Chemical strategies for functional proteomics[J]. Molecular Cellular Proteomics, 2002, 1: 781-790.
[7] Templin M F, Stoll D, Schwenk J M, et al. Protein microarrays: promising tools for proteomic research[J]. Proteomics, 2003, 3: 2155-2166.
[8] Grg A, Weiss W, Dunn M J. Current two-dimensional eletrophoresis technology for proteomics[J]. Proteomics, 2004, 4: 3665-3685.
[9] Lilley K S, Razzaq A, Dupree P. Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation[J]. Current Opinion in Chemical Biology, 2002, 6: 46-50.
[10] Herman E M, Helm R M, Jung R, et al. Genetic modification removes an immunodominant allergen from soybean[J]. Plant Physiology, 2003, 132: 36-43.
[11] Cho J H, Hwang H, Cho M H, et al. The effect of DTT in protein preparations for proteomic analysis: removal of a highly abundant plant enzyme, ribulose bisphosphate carboxylase/oxygenase[J]. Plant Biology, 2008, 51: 297-301.
[12] Shaw M M, Riederer B M. Sample preparation for two-dimensional gel electrophoresis[J]. Proteomics, 2003, 3: 1408-1417.
[13] Gutteridge S, Gatenby A A. Rubisco systhesis, assembly, mechanism, and regulation[J]. Plant Cell, 1995, 7: 809-819.
[14] Corthals G L, Wasinger V C, Hochstrasser D F, et al. The dynamic range of protein expression: a challenge for proteomic research[J]. Electrophoresis, 2000, 21: 1104-1115.
[15] Pier G R, Annalisa C, Ben H. Prefractionation techniques in proteome analysis[J]. Proteomics, 2003, 3: 1397-1407.
[16] Pier G R, Annalisa C, Paolo A. Prefractionation techniques in proteome analysis: The mining tools of the third millennium[J]. Electrophoresis, 2005, 26: 297-319.
[17] Peltier J B, Ripoll D R, Friso G, et al. Clp protease complexes from photosynthetic and non-photosynthetic plastids and mitochondria of plants, their predicted three-dimensional structures, and functional implication[J]. Journal of Biological Chemistry, 2004, 279(6): 4768-4781.
[18] Wienkoop S, Glinski M, Tanaka N, et al. Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins[J]. Rapid Commun Mass Spectrom, 2004, 18: 643-650.
[19] Espagne C, Martinez A, Valot B, et al. Alternative and effective proteomic analysis in Arabidopsis[J]. Proteomics, 2007, 7: 3788-3799.
[20] Kwanyuen P, Allina S M, Weissinger A K, et al. A new form of crystalline rubisco and the conversion too its common dodecahedral form[J]. Joural of Proteome Research, 2002, 1: 471-473.
[21] Widjaja I, Naumann K, Roth U, et al. Combining subproteome enrichment and Rubisco depletion enables identification of low-abundance proteins differentially regulated during plant defense[J]. Proteomics, 2009, 9: 138-147.
[22] Sun T K, Kyu S C, Yu S J, et al. Two-dimensional electrophoresis analysis of rice proteins by polyethylene glycol fractionation for protein arrays[J]. Electrophoresis, 2001, 22: 2103-2109.
[23] Xi J H, Wang X, Li S Y, et al. Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome[J]. Phytochemistry, 2006, 67: 2341-2348.
[24] Lee D G, Ahsan N, Lee S H, et al. An approach toidentify cold-induced low-abundant proteins in rice leaf[J]. Comptes Rendus Biologies, 2007, 330: 215-225.
[25] 苏衍菁, 赵国琦, 王小山, 等. 不同生育期脆性突变水稻细胞壁组分动态研究[J]. 草业学报, 2010, 19(1): 151-157.
[26] 常宏, 王汉宁, 张金文, 等. 玉米品种真实性和纯度鉴定的SSR标记多重PCR体系优化[J]. 草业学报, 2010, 19(2): 204-211.
[27] 邵宏波, 梁宗锁, 邵明安. 小麦抗旱生理生化和分子生物学研究进展与趋势[J]. 草业学报, 2006, 15(3): 5-17.
[28] 惠文森, 穆晓峰, 王康英. 草坪草屑叶蛋白质提取方法研究[J]. 草业科学, 2009, 26(3): 108-110.
[29] Tatsuro H, Alexander E, Ryu O. Gene expression of enzymes for starch ad sucrose metabolism and transport in leaf sheaths of rice (Oryza sativa L.)during the heading period in relation to the sink to source transition[J]. Plant Production Science, 1999, 2: 178-183.
[30] 李兆伟, 熊君, 李振方, 等. 水稻灌浆期叶鞘蛋白质差异表达分析[J]. 作物学报, 2008, 34(4): 619-626.
[31] Kim S T, Cho K S, Jang Y S, et al. Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays[J]. Electrophoresis, 2001, 22: 2103-2109.
[32] Yang P F, Liang Y, Shen S H, et al. Proteome analysis of rice uppermost internodes at the milky stage[J]. Proteomics, 2006, 6: 3330-3338.
[33] Krishnan H B, Natarajan S S. A rapid method for depletion of Rubisco from soybean (Glycine max) leaf for proteomic analysis of lower abundance proteins[J]. Phytochemistry, 2009, 70: 1958-1964.