适用于水稻微粒体蛋白的双向电泳技术及苯达松抗性相关蛋白的初步分析

针对不同来源的样品，建立相应的双向电泳(two-dimensional gel electrophoresis, 2-DE)技术是开展蛋白质组学研究的基础。以野生型水稻(O. sativa L.)农林8号(N8)及其苯达松敏感致死(bentazon sensitive lethal, bsl)突变体农林8号m (N8m)为材料，通过优化条件，建立了适合于水稻微粒体蛋白的双向电泳技术。并通过双向电泳图谱的比对，共找到15个差异蛋白点，分为供试材料间差异、苯达松处理前后差异和N8在苯达松处理后特异表达等3种类型。结合苯达松抗性比较及苯达松残留分析，推测材料间差异是因γ射线辐照引起基因结构变异，从而影响了基因的表达；苯达松处理前后差异与氧自由基清除酶系或系统获得抗性相关酶系有关；N8在苯达松处理后特异表达与苯达松代谢有关。

A Two-dimensional Gel Electrophoresis Protocol Suitable for Rice Microsome Proteins and the Analysis of Bentazon Tolerance-Related Proteins in Rice

Development of an appropriate two-dimensional gel electrophoresis (2-DE) technique for samples from various sources is important for proteomics study. Here, we report a modified 2-DE protocol suitable for rice microsomal proteins by using differential centrifugation in sample preparation, optimizing length and pH range of immobilized pH gradient (IPG) strips, and modifying the electrophoresis program. We used the 2-DE technique to detect the change of rice microsomal proteins expression induced by bentazon in Norin 8 (N8) as well as its bentazon sensitive lethal mutant Norin 8m (N8m). Results showed that 15 differentially expressed protein spots were found after bentazon treatment between or within the two lines. The proteins in these spots were classified into three types. TypeⅠ proteins showed differential expression between the two lines while typeⅡ proteins differed between the treated and untreated samples, and typeⅢ proteins were only induced in N8 after the treatment. TypeⅠ proteins arise from rice DNA damage by γ-ray radiation, based on the fact that N8m is a new strain from N8 mutated by γ-ray. Our previous study indicated that N8 and N8m showed no significant difference in the alteration of photosynthesis in the early stage after bentazon treatment, which induced accumulation of oxidative stress. It can be confirmed that typeⅡ proteins are related to the reactive oxygen scavenging system or systemic acquired resistance. The analysis of bentazon content change in 24 h indicated that N8 could metabolize the absorbed bentazon thoroughly while residual bentazon was present in the treated leaves of N8m. Therefore, we predict the typeⅢ proteins to be related to bentazon metabolism.