Welcome to Acta Agronomica Sinica，

Acta Agron Sin ›› 2010, Vol. 36 ›› Issue (4): 612-619.doi: 10.3724/SP.J.1006.2010.00612

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Construction of Protein Two-Dimensional Polyacrylamide Gel Electrophoresis System for Brassica napus

GAN Lu1,2,LI Dian-Rong1,3,ZANG Xin4,FU Chun-Hua1,2,YU Long-Jiang1,2,LI Mao-Teng12*   

  1. 1 Institute of Resource Biology and Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 43007, China; 2 Key Laboratory of Molecular Biophysics, Ministry of Education, Wuhan 430074, China; 3 Hybrid Rapeseed Research Center of Shaanxi Province, Dali 715105, China; 4 Bioengineering Department of Zhengzhou University, Zhengzhou 450001, China
  • Received:2009-09-08 Revised:2009-12-24 Online:2010-04-12 Published:2010-02-09
  • Contact: LI Mao-Teng, E-mail: limaoteng426@163.com; limaoteng426@mail.hust.edu.cn

PDF

1544

Cited

24

Abstract:

Two-dimensional polyacrylamide gel electrophoresis has been widely applied in proteomic researches. The approach can analyze dynamic changes of proteomics of biological samples under different conditions to identify specific proteins and genes. The two-dimensional gel systems for rice, Arabidopsis and other species have been established. The purpose of this research was to establish a suitable system for B. napus to analyze proteomics. Rapeseed variety of 08127 was used in the experiment, the proteins were obtained by the trichloroacetic extraction method with pH 4–7 gel brands under the improved IEF procedure, showing a clean two-dimensional electrophoresis map, so a proper system for B. napus wasformed. Then the system was validated by using the young seedlings, leaves and stems from other varieties, and the same results as those from variety 08127 were obtained. The proteomics profiling of seedlings in different ages of B. napus was analyzed by using the previous established protocol, we found 20 up-regulated and 51 down-regulated proteins with more than two times changes in 30 days old B. napus stems compared with in 15 days old B. napus stems and some of the different protein spots were successfully identified. The system established in this study is useful for B. napus proteomic research in the future.

Key words: Brassica napus, Protein, Two-dimensional polyacrylamide gel electrophoresis

[1] Choe L H, Lee K H. A comparison of three commercially available isoelectricfocusing units for proteome analysis: The multiphor, the IPGphor and the protean IEF cell. Electrophoresis, 2000, 21: 993–1000

[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] Liang Y(梁宇), Jing Y-X(荆玉祥), Shen S-H(沈世华). Advances in plant proteomics. Acta Phytoecol Sin (植物生态学报), 2004, 28(1): 114–125 (in Chinese with English abstract)

[5] O'Farrell P H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem, 1975, 250: 4007–4021

[6] Bjellqvist B, Ek K, Righetti P G, Gianazza E, Görg A, Westermeier R, Postel W. Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications. J Biochem Biophys Methods, 1982, 6: 317–39

[7] Lei H-L(雷红灵), Fu M(付明), WU Y-Y(吴永尧). Study on two-dimension electrophoresis of seed proteins of Cardamine enshiensis. Hubei Agric Sci (湖北农业科学), 2008, 47(10): 1114–1116 (in Chinese with English abstract)

[8] 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

[9] 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

[10] Rutschow H, Ytterberg A J, Friso G, Nilsson R, van Wijk K J. Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis. Plant Physiol, 2008, 148: 156–175

[11] 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

[12] 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. J Proteome Res, 2008, 7: 1750–1760

[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] 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

[15] Chen S B, Gollop N, Heuer B. Proteomic analysis of salt-stressed tomato (Solanum lycopersicum) seedlings: Effect of genotype and exogenous application of glycinebetaine. J Exp Bot, 2009, 60: 2005–2019

[16] 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)

[17] Meza-Basso L, Alberdi M, Raynal M, Ferrero-Cadinanos M L, Delseny M. Changes in protein synthesis in rapeseed (Brassica napus) seedlings during a low temperature treatment. Plant Physiol, 1986, 82: 733–738

[18] Mihr C, Baumgartner M, Dieterich J H, Schmitz U K, Braun H P. Proteomic approach for investigation of cytoplasmic male sterility (CMS) in Brassica. J Plant Physiol, 2001, 158: 787–794

[19] Hajduch M, Casteel J E, Hurrelmeyer K E, Song Z, Agrawal G K, Thelen J J. Proteomic analysis of seed filling in Brassica napus: Developmental characterization of metabolic isozymes using high-resolution two-dimensional gel electrophoresis1. Plant Physiol, 2006, 141: 32–46

[20] Agrawal G K, Hajduch M, Graham K, Thelen J J. In-depth investigation of the soybean seed-filling proteome and comparison with a parallel study of rapeseed. Plant Physiol, 2008, 148: 504–518

[21] Sheoran I S, Pedersen E J, Ross A R, Sawhney V K. Dynamics of protein expression during pollen germination in canola (Brassica napus). Planta, 2009, 230: 779–793

[22] Desclos M, Dubousset L, Etienne P, Caherec F L, Satoh H, Bonnefoy J, Ourry A, Avice J C. A proteomic profiling approach to reveal a novel role of Brassica napus drought 22 kD/water-soluble chlorophyll-binding protein in young leaves during nitrogen remobilization induced by stressful conditions. Plant Physiol, 2008, 147: 1830–1844

[23] Damerval C, Vienne D D, Zivy M, Thiellement H. Technical improvement in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling protein. Electrophoresis, 1986, 7: 53–54

[24] Gu R-S(谷瑞升), Liu Q-L(刘群录), Chen X-M(陈雪梅), Jiang X-N(蒋湘宁). An improved method of 2D electrophoresis for protein analysis of woody plants. J Beijing For Univ (北京林业大学学报–10 (in Chinese with English abstract)), 1999, 21(5): 7

[25] Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri G M, Carnemolla B, Orecchia P, Zardi L, Righetti P G. Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis,2004, 25: 1327–1333

[26] Li X H, Wu X F, Yue W F, Liu J M, Li G L, Miao Y G. Proteomic analysis of the silkworm (Bombyx mori L.) hemolymph during developmental stage. J Proteome Res, 2006, 5: 2809–2814

[27] Ruan S-L(阮松林), Ma H-S(马华升), Wang S-H (王世恒), Xin Y(忻雅), Qian L-H(钱丽华), Tong J-X(童建新), Zhao H-P(赵杭苹), Wang J(王杰). Adevances in plant proteomics I: Key techniques of proteome. Hereditas (遗传), 2006, 28(11): 1472–1486 (in Chinese with English abstract)

[28] Garfin D. Two-dimensional gel electrophoresis: An overview. Trends Anal Chem, 2003, 22: 263–272

[29] Wu M-C(吴满成), Hu H-T(胡海涛), Yu Y-J(余有见), Sun N(孙娜), Yang L(杨玲). Extraction and improvement of two-dimensional electrophoresis analysis of proteins form berries of Elaeagnus umbellate Thunb. Plant Physiol Commun (植物生理学通讯), 2009, 45: 695–698 (in Chinese)
[1] CHEN Song-Yu, DING Yi-Juan, SUN Jun-Ming, HUANG Deng-Wen, YANG Nan, DAI Yu-Han, WAN Hua-Fang, QIAN Wei. Genome-wide identification of BnCNGC and the gene expression analysis in Brassica napus challenged with Sclerotinia sclerotiorum and PEG-simulated drought [J]. Acta Agronomica Sinica, 2022, 48(6): 1357-1371.
[2] WANG Wang-Nian, GE Jun-Zhu, YANG Hai-Chang, YIN Fa-Ting, HUANG Tai-Li, KUAI Jie, WANG Jing, WANG Bo, ZHOU Guang-Sheng, FU Ting-Dong. Adaptation of feed crops to saline-alkali soil stress and effect of improving saline-alkali soil [J]. Acta Agronomica Sinica, 2022, 48(6): 1451-1462.
[3] ZHU Zheng, WANG Tian-Xing-Zi, CHEN Yue, LIU Yu-Qing, YAN Gao-Wei, XU Shan, MA Jin-Jiao, DOU Shi-Juan, LI Li-Yun, LIU Guo-Zhen. Rice transcription factor WRKY68 plays a positive role in Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae [J]. Acta Agronomica Sinica, 2022, 48(5): 1129-1140.
[4] JIN Min-Shan, QU Rui-Fang, LI Hong-Ying, HAN Yan-Qing, MA Fang-Fang, HAN Yuan-Huai, XING Guo-Fang. Identification of sugar transporter gene family SiSTPs in foxtail millet and its participation in stress response [J]. Acta Agronomica Sinica, 2022, 48(4): 825-839.
[5] YUAN Da-Shuang, DENG Wan-Yu, WANG Zhen, PENG Qian, ZHANG Xiao-Li, YAO Meng-Nan, MIAO Wen-Jie, ZHU Dong-Ming, LI Jia-Na, LIANG Ying. Cloning and functional analysis of BnMAPK2 gene in Brassica napus [J]. Acta Agronomica Sinica, 2022, 48(4): 840-850.
[6] XU Jing, GAO Jing-Yang, LI Cheng-Cheng, SONG Yun-Xia, DONG Chao-Pei, WANG Zhao, LI Yun-Meng, LUAN Yi-Fan, CHEN Jia-Fa, ZHOU Zi-Jian, WU Jian-Yu. Overexpression of ZmCIPKHT enhances heat tolerance in plant [J]. Acta Agronomica Sinica, 2022, 48(4): 851-859.
[7] HUANG Cheng, LIANG Xiao-Mei, DAI Cheng, WEN Jing, YI Bin, TU Jin-Xing, SHEN Jin-Xiong, FU Ting-Dong, MA Chao-Zhi. Genome wide analysis of BnAPs gene family in Brassica napus [J]. Acta Agronomica Sinica, 2022, 48(3): 597-607.
[8] WANG Rui, CHEN Xue, GUO Qing-Qing, ZHOU Rong, CHEN Lei, LI Jia-Na. Development of linkage InDel markers of the white petal gene based on whole-genome re-sequencing data in Brassica napus L. [J]. Acta Agronomica Sinica, 2022, 48(3): 759-769.
[9] YANG Zong-Tao, LIU Shu-Xian, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane ubiquitin-like protein UBL5 responses to SCMV infection and interacts with SCMV-6K2 [J]. Acta Agronomica Sinica, 2022, 48(2): 332-341.
[10] ZHAO Gai-Hui, LI Shu-Yu, ZHAN Jie-Peng, LI Yan-Bin, SHI Jia-Qin, WANG Xin-Fa, WANG Han-Zhong. Mapping and candidate gene analysis of silique number mutant in Brassica napus L. [J]. Acta Agronomica Sinica, 2022, 48(1): 27-39.
[11] WANG Na, BAI Jian-Fang, MA You-Zhi, GUO Hao-Yu, WANG Yong-Bo, CHEN Zhao-Bo, ZHAO Chang-Ping, ZHANG Ling-Ping. Cloning and expression analysis of lncRNA27195 and its target gene TaRTS in wheat (Triticum aestivum L.) [J]. Acta Agronomica Sinica, 2021, 47(8): 1417-1426.
[12] ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, LIU Shu-Xian, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane PsbR subunit response to SCMV infection and its interaction with SCMV-6K2 [J]. Acta Agronomica Sinica, 2021, 47(8): 1522-1530.
[13] WANG Yan-Hua, LIU Jing-Sen, LI Jia-Na. Integrating GWAS and WGCNA to screen and identify candidate genes for biological yield in Brassica napus L. [J]. Acta Agronomica Sinica, 2021, 47(8): 1491-1510.
[14] GAO Lu, XU Wen-Liang. GhP4H2 encoding a prolyl-4-hydroxylase is involved in regulating cotton fiber development [J]. Acta Agronomica Sinica, 2021, 47(7): 1239-1247.
[15] LI Jie, FU Hui, YAO Xiao-Hua, WU Kun-Lun. Differentially expressed protein analysis of different drought tolerance hulless barley leaves [J]. Acta Agronomica Sinica, 2021, 47(7): 1248-1258.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No.12 Zhongguancun South Street, Beijing 100081,China Email:xbzw@chinajournal.net.cn
Supported by Beijing Magtech Co.Ltd