巴西橡胶树胶乳橡胶粒子凝集和去凝集的生化调控机制

The natural rubber that is biosynthesized and stored in laticifers of rubber trees meets the most demands for natural rubber in the world. The latex (the resource of refining natural rubber) flows out of the severed laticifers after the trunk bark being cut. The latex flow lasts for several times and thereafter terminates as the result of the plug formation at the end of the severed laticifers. Considering that duration of latex flow is a key factor that directly determines the rubber yield per tapping, the mechanism of laticifer plugging is a major theoretical subject that is faced by the natural rubber production. We have discovered that latex allergen Hev b7 is effective in inhibiting rubber particle aggregation and the ethrel-caused prolongation of the duration of latex flow is associated with the increase in the level of the Hev b7 that hinders rubber particle aggregation. It is so far unknown about the biochemical mechanism for the antagonistic effect of allergen protein Hev b7 on rubber particle aggregating factors. This project is to identify the target protein on the rubber particles that could interact with the promoting and inhibiting factors of rubber particle aggregation, the interact region and major amino acid within the region as well as the interaction between the promoting factors or inhibiting factors by using the biochemical techniques such as protein isolation and purification, preparation of mutant proteins, yeast-2-hybridization, SPR and Pulldown, so as to reveal the regulating mechanism for rubber particle aggregation and deaggregation. The results are also significant for improving the latex flow traits.

橡胶树乳管组织合成和贮存的天然橡胶是全球所需天然橡胶的主要来源。通过切割树干树皮，使胶乳（提炼天然橡胶的原料）从乳管伤口处排出。在排胶持续一定时间后，乳管伤口被堵塞，导致排胶停止。由于排胶持续时间是直接决定天然橡胶产量的关键因素，所以，乳管伤口堵塞机制是天然橡胶生产面临的一个重大理论问题。我们发现胶乳过敏原蛋白Hev b7是有效的橡胶粒子去凝集因子，乙烯利刺激显著延长排胶时间主要是通过增加Hev b7含量而延缓了橡胶粒子的凝集。目前对Hev b7拮抗橡胶粒子凝集因子的生化机制仍不清楚。本项目拟采取蛋白质分离纯化、突变体蛋白制备以及酵母双杂交、SPR和Pulldown等蛋白质互作分析相关技术，鉴定橡胶粒子上与橡胶粒子凝集促进因子和抑制因子结合的靶标蛋白及其互作区段和主要位点以及促进因子或抑制因子之间的互作分析，揭示橡胶粒子凝集和去凝集的调控机制，对于改良橡胶树的排胶性状也具有重要的指导意义。

巴西橡胶树（简称橡胶树）是世界所需天然橡胶主要来源。乳管是橡胶树合成和储存天然橡胶场所。在天然橡胶生产中，通过切割树干树皮（割胶），切断树皮中的乳管，收集从乳管中流出的胶乳，用作提炼天然橡胶的原料。在排胶过程中，堵塞物逐渐在乳管伤口形成和积累，导致排胶速度降低和排胶停止，影响到排胶持续时间。排胶持续时间是决定天然橡胶产量的一个关键因素。橡胶粒子是堵塞物的主要成分，橡胶粒子的凝集和去凝集直接关系到堵塞物的形成。本项目采用柱层析对促进和抑制橡胶粒子凝集的蛋白质进行分离纯化，采用酵母双杂交、荧光素酶互补实验等蛋白质互作分析技术，在蛋白质互作层面揭示橡胶粒子凝集和去凝集的调控机制，取得以下主要结果：（1）促进橡胶粒子凝集的蛋白质有β-1,3-葡聚糖酶GLU、橡胶素HEV、几丁质酶和3-磷酸甘油醛脱氢酶GAPDH等四种，其中，β-1,3-葡聚糖酶GLU1不仅能与橡胶粒子上的小橡胶粒子膜蛋白SRPP1互作，而且与3-磷酸甘油醛脱氢酶GAPDH12互作；橡胶素HEV1和几丁质酶CHI1也能够与3-磷酸甘油醛脱氢酶GAPDH12互作；GLU1/2之间、GLU1和HEV1之间、CHI1和HEV1之间以及CHI1之间有不同程度互作。（2）虽然与抑制橡胶粒子凝集有关的蛋白只有一种Hev b7，但是该蛋白不仅能与SRPP1互作，而且与所有的4种促进因子蛋白互作。（3）进一步分析了这些互作蛋白的互作区段。（4）在强割和乙烯利刺激条件下，橡胶树的排胶时间显著延长，与Hev b7显著增加密切相关。根据这些结果，Hev b7通过与GLU1竞争结合SRPP1以及与促进因子蛋白互作，抑制促进因子蛋白对橡胶粒子的凝集效应，进而延缓堵塞物的形成。本研究结果揭示了橡胶粒子凝集和去凝集的一种新的调控机制，对于改良橡胶树的产量性状具有重要的指导意义。