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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2010, Vol. 4 Issue (4) : 416-421     DOI: 10.1007/s11703-010-1031-9
RESEARCH ARTICLE |
Protein extraction from rice (Oryza sativa L.) root for two-dimensional electrophresis
Xiaoliang XIANG1, Shuju NING2, Xia JIANG3, Xiaogui GONG1, Renlei ZHU1, Lanfang ZHU1, Daozhi WEI1()
1. School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. School of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3. College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350002, China
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Abstract  

An efficient protein extraction method for two-dimensional gel electrophoresis (2-DE) from plant samples is usually challenging due to the low protein content and high level of interfering compounds. Proteomic analyses of rice (Oryza sativa L.) roots are limited by the lack of an efficient protein extraction method. To establish an effective protocol of protein extraction suitable for 2-DE analysis in rice roots, we evaluated three protein extraction methods (trichloroacetic acid [TCA]/acetone, Mg/NP-40/TCA, and tris-base/acetone). Our results showed that the Mg/NP-40/TCA extraction method had the highest protein yield and is the best resolution of protein separation among the three methods. The TCA/acetone method exhibited clear ?protein? profiles? and ?detected? more ?protein ?spots with the highest intensity in the region of high Mr (above 45 kDa) than the other methods. However, this method was unable to detect proteins with low-Mr (less than 24.0 kDa). The Tris-base/acetone method showed the poorest resolution of protein separation. Our results suggest that the Mg/NP-40/TCA method was the most effective among the three methods and may provide enhanced proteomic information for rice and other crop roots.

Keywords protein extraction      two-dimensional electrophresis (2-DE)      rice (Oryza sativa L.) roots     
Corresponding Authors: WEI Daozhi,Email:weidz888@sohu.com   
Issue Date: 05 December 2010
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-010-1031-9     OR     http://academic.hep.com.cn/fag/EN/Y2010/V4/I4/416
methodsprotein yield/(mg·g-1fresh wt)spots number
Mg/NP-40/TCA0.60±0.004757±3.6
TCA/acetone0.33±0.014784±3.2
Tris-base/acetone0.30±0.002708±27.0
Tab.1  Protein yield and total number of spots using different protein extraction methods
Fig.1  SDS-PAGE analysis of rice root proteins extracted with three extractional methods
Note: The proteins were analyzed on 10% SDS-PAGE. Lane M, protein molecular mass standards. Lane A, Lane B, and Lane C: methods A-C). A 15 g quantity of protein was loaded in each lane .The gel was stained with CBB G250 (Blue silver).
Fig.2  Comparison of two-dimensional gel electrophoresis protein pattern in roots of rice ( L.) using different methods
Note: (A): Mg/NP-40/TCA; (B): TCA/acetone; (C): Tris-base/acetone. Proteins (200 g) were separated on a 3-10 nonlinear pH gradient strips and then in the second dimension using 10.0% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were stained with CBB G250.
Fig.3  Scatter plots of 2-DE analysis of total proteins extracted from rice roots by three methods
Note: The gel maps were analyzed by Image Master 2D Platinum Trial software taking the volume fraction as the spot value type, and the -axis indicates the abundance of the proteins from one protein extraction method 2-DE map, and the -axis indicates those from the other protein extraction method 2-DE map.
Fig.4  Quantitative analysis of some selected significant spot region from three methods in 2-DE gels
Note: 1, 2, 3, 4: Example of a spot region that is significantly different in Fig. 2. Protein spots 3D views analyzed by the Image Master 2D Platinum Trial software. Spot region numbers correspond with the number in Fig.2.
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