橡胶粒子膜蛋白双向电泳体系的建立和质谱初步分析

橡胶粒子是橡胶树乳管细胞中进行橡胶生物合成的一种特殊细胞器。参与橡胶生物合成的橡胶转移酶、橡胶延长因子等重要酶类和相关的蛋白质调控因子均定位于橡胶粒子的表面或镶嵌于橡胶粒子膜中。本研究在SDS-PAGE分离橡胶粒子膜蛋白的基础上,使用pH3￣10非线性IPG胶条进行2-DE双向电泳,成功地分离了橡胶粒子膜蛋白,在2-DE银染胶上共得到约520个蛋白质点,并通过基质辅助激光解吸附飞行质谱(MALDI-TOF)对部分蛋白质点进行了初步分析鉴定。检索蛋白质数据库证明其中的蛋白质点包括橡胶延长因子(REF)和橡胶过敏蛋白Hev3等典型的橡胶粒子膜蛋白,另外还有一些功能未知的蛋白质。从银染2-DE胶上可以看出,REF在橡胶粒子膜蛋白中占有很大的比例,这表明REF在橡胶生物合成中可能具有非常重要的作用。通过比较蛋白质组学研究发现,橡胶粒子膜上存在乙烯和茉莉酸信号响应蛋白。乙烯和茉莉酸对橡胶生物合成的调控作用可能与橡胶粒子膜上存在它们的响应蛋白有关。橡胶粒子膜蛋白2-DE双向电泳体系的建立为全面研究橡胶粒子膜蛋白的组成及其变化奠定了基础。

Establishment of 2-DE System and Primary Analyses on the Membrane Proteins of Rubber Particles in Hevea brasiliensis by MALDI-TOF

Rubber particles are important organelles in latex for rubber biosynthesis in rubber tree (Hevea brasilensis). The crucial enzymes in rubber biosynthesis such as rubber transferase, rubber elongation factor and other proteins and so on were all located at the surface or embedded in the membranes of rubber particles. In this study, the membrane proteins of rubber particles had been successfully isolated, and further examined by SDS- PAGE (10 % w/v gel). Afterwards, 2-DE was performed using the nonlinear gel strip pH3-10 to separate the membrane proteins. About 520 protein spots were obtained on the silver stained gel. Some protein spots were taken randomly and analyzed by MALDI-TOF. Through searching the protein database and blast, some previously known proteins were further identified including the rubber elongation factor (REF), rubber allergic protein Hev 3, etc, and some were unknown proteins. From the silver stained gel, it could be seen that the REF contributed substantially to the membrane proteins of rubber particles, indicating that REF might have a very important function in the rubber biosynthesis. JA and ET responsive proteins were also found in the membrane proteins, which implied that JA and ET might regulate rubber biosynthesis through these responsive proteins on the membranes of rubber particles in the rubber tree. The establishment of the 2-DE system on the rubber particles is conducive to investigating the components and changes of rubber particles membrane proteins.