橡胶树不同粒径橡胶粒子结合凝集相关蛋白的分析

橡胶树是重要的产胶植物，其树皮组织中的次生乳管是合成和贮存天然橡胶的主要组织。天然橡胶是从乳管中的胶乳中提炼而成，由特殊的细胞器-橡胶粒子合成。橡胶粒子为半个单位膜的球状结构，内部贮存合成的天然橡胶，外周膜上结合有多种蛋白质，这些蛋白与其执行的功能密切相关。目前对天然橡胶合成相关蛋白的研究较多，对与橡胶粒子凝集相关的蛋白研究甚少。以‘RY7-33-97’胶乳为材料，利用不同的离心速度，分级分离获取不同粒径的橡胶粒子，并进行不同程度的清洗，以Tricine-SDS-PAGE以及Western-blotting技术对橡胶粒子上结合的蛋白进行比较分析。研究结果表明，不同粒径的橡胶粒子上结合的蛋白含量存在差异。小橡胶粒子膜蛋白（SRPP）随着粒径的减小含量明显增加；而橡胶延伸因子蛋白（REF）的含量基本维持稳定，与粒径大小关系不大。存在于C-乳清中的Hev b7胶乳过敏原蛋白和3-磷酸甘油醛脱氢酶（GAPDH）与橡胶粒子有明显的结合；存在于黄色体B-乳清中的β-1,3-葡聚糖酶（Glu）、橡胶素（Hev）和几丁质酶（Chit）均能与橡胶粒子结合，但几丁质酶与之的结合能力最弱。橡胶粒子经清洗后，作为橡胶粒子的主要膜蛋白，SRPP含量随着清洗次数增加明显降低，而REF蛋白含量变化不明显，可见SRPP与橡胶粒子膜的结合紧密程度不如REF；来自C-乳清和B-乳清的凝集相关蛋白随清洗次数增加也明显降低含量。体外孵育小橡胶粒子与不同的蛋白样品，结果表明，橡胶粒子可体外结合多种不同的蛋白，其中多种蛋白都与橡胶粒子的凝集有关。本研究对不同粒径橡胶粒子上结合的凝集相关蛋白进行了初步解析，旨在为阐明橡胶树排胶机制奠定基础。

Aggregation-related Proteins Binding to Rubber Particles with Different Diameter in Rubber Tree

Rubber tree (Hevea brasiliensis Muell.Arg.) is an important rubber-producing plant, and the secondary laticifer in the trunk bark is the major tissue for the synthesis and storage of natural rubber. Natural rubber is extracted from the latex in laticifer by tapping (mechanical wounding) and synthesized by a special organelle, named the rubber particle. Rubber particle is a spherical structure surrounded by a lipid monolayer and membrane-bound proteins, in which the natural rubber is stored. The peripheral membrane is bound to a variety of proteins that are closely related to the functions of rubber particles. At present, there are many studies on proteins related to natural rubber synthesis, but the proteins related to rubber particle aggregation still remains unclear. Using the latex collected from the clone ‘RY7-33-97’ as the material, rubber particles with different particle sizes were obtained by fractionation at different centrifugal speeds. Subsequently, the rubber particle samples were cleaned by buffer for three times and collected for electrophoresis, respectively. The binding proteins on rubber particles were compared and analyzed by Tricine- SDS-PAGE and Western-blotting techniques. There were obvious differences in the content of bound proteins on rubber particles with different particle size. Accompanied by the decrease of rubber particle size, the content of small rubber particle membrane protein (SRPP) increased significantly, while the content of rubber elongation factor protein (REF) remained stable and had little relationship with the rubber particle size. Hevb7 latex allergen protein (Hevb7) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in C-serum could bind to the rubber particles, and β-1,3-glucanase (Glu), hevein (Hev) and chitinase (Chit) in B-serum could also bind to rubber particles, but in which the binding ability of chitinase to rubber particles was the weakest. After cleaning, as the main membrane protein of rubber particles, the content of SRPP decreased significantly with the times of cleaning, but the content of REF protein did not change obviously, indicating that the binding ability of SRPP to rubber particle membrane is not as strong as that of REF. The content of agglutination-related proteins from C-serum and B-serum decreased significantly with the times of cleaning, usually, the proteins could not be detected after the second cleaning. Small rubber particles with about 0.16 μm mean diameter were incubated with different protein samples in vitro, the results showed that rubber particles could bind a variety of different proteins, in which some proteins were related to the aggregation of rubber particles. In this study, the aggregation-related proteins bound to rubber particles with different particle sizes were preliminarily analyzed, the results would lay a foundation for elucidating the mechanism of latex flow of rubber trees.