《植物生理学报》 2014, 50(10): 1601-1607

甘蓝型油菜温敏核不育系SP2S花蕾总蛋白质双向电泳体系的建立及应用

张菁雯, 李晓荣, 赵燕妮, 胡玉梅, 徐献锋, 于澄宇*

西北农林科技大学农学院, 旱区作物逆境生物学国家重点实验室, 陕西杨凌712100

通信作者：于澄宇；E-mail: yu1009@nwsuaf.edu.cn

摘　要：

以甘蓝型油菜温敏核不育系SP2S及其近等基因系SP2F为材料, 分别采用TCA/丙酮法和Tris-丙酮-酚法提取花蕾总蛋白, 并优化电泳体系, 对可育和不育花蕾中的蛋白质表达进行差异分析。结果表明: TCA/丙酮法适合于花蕾总蛋白的提取, 采用10%浓度的分离胶, 上样量为450 μg, 用pH 4~7的17 cm线性胶条和改良的考马斯亮蓝染色法得到了背景清晰、蛋白点分布均匀且重复性好的电泳凝胶图谱。利用这一实验体系, 筛选出SP2S和SP2F之间大量的差异蛋白点。相比SP2F, 仅在SP2S中表达的有13个点, 上调2倍以上的点有4个, 下调2倍以上的点有9个。

关键词：甘蓝型油菜; 温敏核不育; 花蕾; 双向电泳; 蛋白质

收稿：2014-06-19   修定：2014-09-25

资助：教育部新世纪优秀人才项目(NECT-10-0693)和现代农业产业技术体系(ARS-13)。

Establishment and Application of Two-Dimensional Electrophoresis System for Floral Bud Proteome of Temperature-Sensitive Male Sterile Line SP2S in Brassica napus

ZHANG Jing-Wen, LI Xiao-Rong, ZHAO Yan-Ni, HU Yu-Mei, XU Xian-Feng, YU Cheng-Yu*

College of Agronomy, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest Agriculture and Forestry University, Yangling, Shaanxi 712100, China

Corresponding author: YU Cheng-Yu; E-mail: yu1009@nwsuaf.edu.cn

Abstract:

Using TCA/acetone and Tris-acetone-phenol methods to extract total proteins from floral buds respectively, an optimized system of two-dimensional electrophoresis (2-DE) was employed to screen differential protein expression between a temperature-sensitive male sterile line SP2S and its near isogenic line SP2F in Brassica napus. The results showed that clear background, well distributed protein spots and reproducible 2-DE maps could be acquired following TCA/acetone extraction, electrophoresis system including 10% SDS-PAGE, 450 μg of sample loading quantity, and 17 cm immobilized pH gradient (IPG) strip with pH 4–7, and modified staining using Coomassie Brilliant Blue. Using this optimized 2-DE system some differential protein spots between SP2S and SP2F were found. Compared with SP2F, 13 spots were specifically expressed in SP2S, 4 of them were up-regulated more than 2-fold and 9 were down-regulated more than 2-fold.

Key words: Brassica napus; temperature-sensitive male sterility; floral bud; two-dimensional electrophoresis; protein