巴西橡胶树乳管伤口堵塞物形成机制的研究

【作者】 史敏晶； 田维敏；

【机构】 中国热带农业科学院橡胶研究所；

【摘要】 排胶时间是限制天然橡胶产量的关键因素之一,而乳管伤口堵塞物的形成是决定排胶时间的主要因子,因此,乳管伤口堵塞机制一直是天然橡胶生产中面临的一个重大理论问题。以往人们一直认为乳管堵塞是乳管伤口处胶乳凝固(即橡胶粒子凝固形成橡胶凝块)的结果,其机制与胶乳体外凝固是一样的。最近,我国学者采用组织化学和电镜技术研究了刚停止排胶时的乳管伤口堵塞物的性质,发现乳管伤口堵塞物由蛋白质性质的网状结构("蛋白质网")和橡胶粒子的聚集物组成,没有观察到橡胶凝块,表明乳管伤口最初的堵塞与橡胶凝块形成无关。但是,乳管伤口堵塞物有哪些成分?蛋白质网的成分及其交链机制怎样?橡胶粒子与"蛋白质网"是怎样相互作用的?等诸多问题仍有待阐明。本文采用细胞学、生物化学和蛋白质组学相关技术,较深入地研究了乳管伤口堵塞物形成的机制,结果如下:1、优化了黄色体β-乳清可溶性蛋白质电泳分离技术、免疫印迹技术和间接免疫荧光定位技术;2、蛋白质网的主要成分是来自黄色体的橡胶素、几丁质酶和β-1,3-葡聚糖酶这三种蛋白质内含物;3、参与蛋白质网组成的β-1,3-葡聚糖酶是一种碱性的β-1,3-葡聚糖酶,分子量分别为35.295KDa和41.543KDa,等电点为9.4左右。在接近胶乳生理pH条件下,它们能紧密结合黄色体膜;4、在接近胶乳生理pH条件下,橡胶素和β-1,3-葡聚糖酶之间以及几丁质酶和前橡胶素及其C端蛋白质之间相互作用;5、橡胶粒子主要通过橡胶素结合到蛋白质网上,而黄色体膜主要通过β-1,3-葡聚糖酶结合到蛋白质网上。在我们的研究结果和已有工作的基础上,我们提出一个较为完善的乳管伤口堵塞物形成的模型,主要内容包括:(1)以橡胶素、几丁质酶和β-1,3-葡聚糖酶为主要组分的蛋白质网在堵塞物的形成中起核心作用;(2)橡胶素和β-1,3-葡聚糖酶、几丁质酶和前橡胶素及其C-端蛋白质通过物理作用相交联是蛋白质网形成的生化基础;(3)黄色体膜碎片通过与β-1,3-葡聚糖酶的结合交联到蛋白质网上;(4)橡胶粒子主要通过与橡胶素的结合聚集到蛋白质网上,直到完全停排,橡胶粒子仍然保持膜的完整性;(5)黄色体和C-乳清中pH的差异可能是堵塞物形成的生理基础;(6)乳管伤口的堵塞物不仅有阻止胶乳从乳管中流失的作用,而且具有防御病原物入侵伤口的功能。研究结果为进一步研究乳管伤口堵塞物形成的调节机制打下了良好基础,为开发安全有效的排胶刺激剂提供了理论依据。更多还原

【Abstract】 Duration of latex flow after tapping is one of the three critical factors that limits rubber yield, which is mainly determined by the formation of plug materials at the end of laticifer cut. For this reason, the mechanism by which the wounded laticifers are plugged has been a fundamentally theoretical barrier that challenges natural rubber production. It has been so far generally accepted that the obstruction and stopping of latex flow after tapping is attributed to the formation of coagula from rubber particles and this process is the same in principle as that occuring in vitro. In contrast to the conception, Hao et al. (2004) found that after tapping, the laticifer wounds were not plugged by the rubber particle coagula but by a protein-network, together with rubber particle aggregations by using light-and electron-microscopy. Now such questions as what is the components of the plug materials, what is the components of the protein-network and how are they cross-linked, and how do the rubber particles interact with the protein-network are still remained to be answered. In this paper, we investigated in more detail the mechanism by which the plug materials formed by means of cytological, biochemical and proteomic techniques. The results as follows:1. We optimized the conditions of SDS-PAGE, Western-blot and immuno-histochemical localization for the analysis of proteins from B-serum in lutoids;2. The major components of the protein-network were hevein, chitinase and β-1,3-glucanase, which are the main three proteins of the protein inclusions in lutoids; 3. The β-1,3-glucanases associated with protein-network was of a basic type with molecular weight about 35.295-and 41.543 ku and pI of about 9.4. They combined closely with lutoid membrane at the pH6.9 which is close to the physiological pH value of latex.4. Interactions occurred between hevein and β-1,3-glucanase, and between chitinase and hevein-precursor as well as its carboxyl-terminal domain under the condition of the physiological pH value of latex.5. Rubber particles were absorbed to the protein-network by combination with hevein while the lutoid membrane fragments with β-1,3-glucanase. On the basis of the available evidences, we conclusively make a model for elucidating the mechanism by which laticifer plug formation. The main contents of this model are: (1) Protein-network which is consist of hevein, chitinase and β-1,3-glucanase is essential for laticifer plug formation; (2) The biochemical bases for protein-network formation are the interactions between hevein and β-1,3-glucanase, and between chitinase and hevein-precursor as well as its carboxyl-terminal domain under the condition of the physiological pH value of latex.; (3) Lutoid membrane fragments were absorbed to the protein-network by combination with β-1,3-glucanase; (4) Rubber particles were absorbed to the protein-network by combination with hevein and remain intact until latex flow completely stops; (5) The physiological basis for laticifer plug formation may be the difference in pH values between lutoids and C-serum; (6) The laticifer plugs may play roles in both keeping latex in laticifers and preventing wounded laticifers from pathogen invasion. The results will facilitate the investigation of the mechanism for the regulation of laticifer plug formation and exploitation of some safe and more efficient stimulus, which is necessary for rubber production.更多还原