欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2010, Vol. 36 ›› Issue (4): 612-619.doi: 10.3724/SP.J.1006.2010.00612

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜蛋白质双向电泳体系的建立

甘露1,2，李殿荣1,3，臧新4，付春华1,2，余龙江1,2，栗茂腾1,2,*   

  1. 1华中科技大学生命科学与技术学院,湖北武汉430074;2分子生物物理教育部重点实验室,河北武汉430074;3陕西省杂交油菜研究中心,陕西大荔715105;4郑州大学生物工程系,河南郑州450001
  • 收稿日期:2009-09-08 修回日期:2009-12-24 出版日期:2010-04-12 网络出版日期:2010-02-09
  • 通讯作者: 栗茂腾, E-mail: limaoteng426@163.com; limaoteng426@mail.hust.edu.cn
  • 基金资助:
    本研究由国家重点基础研究发展计划(863计划)项目(2009AA101105)，新世纪优秀人才支持计划和华中科技大学自主创新基金(M2009059)项目资助。

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 Published:2010-04-12 Published online:2010-02-09
  • Contact: LI Mao-Teng, E-mail: limaoteng426@163.com; limaoteng426@mail.hust.edu.cn

PDF

1542

被引次数

54

摘要：

选用油菜品种08127，采用TCA-丙酮提取方法，pH 4~7胶条和改进的IEF聚焦程序，得到较好的蛋白质双向电泳结果，建立了一种适合甘蓝型油菜的双向电泳体系；利用其他甘蓝型油菜品种的幼苗、叶片和茎中的蛋白检验了建立的实验体系，都能得到较好的蛋白质双向电泳结果；利用该实验体系比较甘蓝型油菜幼苗不同发育时期蛋白质双向电泳图，找到二者之间的差异点，选取部分点成功进行了质谱分析。

关键词: 甘蓝型油菜, 蛋白质, 双向电流

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] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501.
[3] 朱峥, 王田幸子, 陈悦, 刘玉晴, 燕高伟, 徐珊, 马金姣, 窦世娟, 李莉云, 刘国振. 水稻转录因子WRKY68在Xa21介导的抗白叶枯病反应中发挥正调控作用[J]. 作物学报, 2022, 48(5): 1129-1140.
[4] 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850.
[5] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[6] 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769.
[7] 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510.
[8] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[9] 唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990.
[10] 周新桐, 郭青青, 陈雪, 李加纳, 王瑞. GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状[J]. 作物学报, 2021, 47(4): 587-598.
[11] 李书宇, 黄杨, 熊洁, 丁戈, 陈伦林, 宋来强. 甘蓝型油菜早熟性状QTL定位及候选基因筛选[J]. 作物学报, 2021, 47(4): 626-637.
[12] 张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659.
[13] 唐婧泉, 王南, 高界, 刘婷婷, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. 甘蓝型油菜SnRK基因家族生物信息学分析及其与种子含油量的关系[J]. 作物学报, 2021, 47(3): 416-426.
[14] 蒙姜宇, 梁光伟, 贺亚军, 钱伟. 甘蓝型油菜耐盐和耐旱相关性状的QTL分析[J]. 作物学报, 2021, 47(3): 462-471.
[15] 魏丽娟, 申树林, 黄小虎, 马国强, 王曦彤, 杨怡玲, 李洹东, 王书贤, 朱美晨, 唐章林, 卢坤, 李加纳, 曲存民. 锌胁迫下甘蓝型油菜发芽期下胚轴长的全基因组关联分析[J]. 作物学报, 2021, 47(2): 262-274.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2