《植物生理学报》 2012, 48(3): 303-309

甜瓜叶片中Rubisco的去除及双向电泳体系的优化

钟俐, 马成梅, 梁永增, 李冠*

新疆大学生命科学与技术学院, 乌鲁木齐830046

通信作者：李冠；E-mail: guanli@xju.edu.cn；Tel: 0991-8581106

摘　要：

为降低Rubisco的干扰, 建立适合于甜瓜叶片蛋白质的双向电泳技术, 本文比较了不同全蛋白提取方法和上样量对双向电泳的影响。结果表明, Mg/NP-40/PEG3350/TCA/丙酮提取法可去除样品中绝大多数的Rubisco, 使低丰度蛋白得以检测, 适合后续分析。以该法提取的蛋白, 采用600、800、1 000和l 200 μg四种上样量进行双向电泳, 上样量为l 000 μg的样品用pH 3~10的IPG胶条, 在2-DE胶上分辨出质量好、数量多的蛋白质点(562个)。因此, Mg/NP-40/PEG3350/TCA/丙酮法是适合甜瓜叶片蛋白质提取的方法, l 000 μg是最适上样量。

关键词：甜瓜; Rubisco; 蛋白质提取; 双向电泳; 上样量

收稿：2011-12-12   修定：2012-01-20

资助：国家自然科学基金(31060148)、 新疆维吾尔自治区自然科学基金(2011211A013)和新疆维吾尔自治区高技术研究发展项目(201111120)。

Depletion of Rubisco from Cucumis melon Leaf and Optimization of Two-Dimensional Electrophoresis for Proteome

ZHONG Li, MA Cheng-Mei, LIANG Yong-Zeng, LI Guan*

College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

Corresponding author: LI Guan; E-mail: guanli@xju.edu.cn; Tel: 0991-8581106

Abstract:

This research compared the effects of different methods of protein extraction of Cucumis melon leaves and different loading amounts on 2-DE profile. The results indicated that Mg/NP-40/PEG3350/TCA/acetone precipitation was a suitable method for subsequent analysis attributed to reducing the interference of Rubisco. There were 562 proteins could be well resolved and detected in the gels when l 000 μg protein of leaves was loading with pH 3-10 IPG strips. All the results showed that Mg/NP-40/PEG3350 TCA/acetone precipitation was much more suitable for protein extraction and l 000 μg was the optimum loading amount of Cucumis melon leaves.

Key words: Cucumis melo L.; Rubisco; protein extraction; two-dimensional electrophoresis (2-DE); loading quantity of sample