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

doi:10.3969/j.issn.1000-2561.2022.12.003

Abstract

Abstract:

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.

Key words: Hevea brasiliensis Muell.Arg., rubber particle, aggregation-related protein, western blotting

CLC Number:

S794.1

DING Huan, WU Shaohua, XIA Zhihui, HUANG Xi, SHI Minjing. Aggregation-related Proteins Binding to Rubber Particles with Different Diameter in Rubber Tree[J]. Chinese Journal of Tropical Crops, 2022, 43(12): 2413-2421.

Figures/Tables 4

Fig. 1 Ratio of rubber particles with different diameters in fresh latex collected by different centrifugation strength

Fig. 2 Analysis of protein binding to the rubber particles with different diameter 1-5: The samples with 0.45, 0.37, 0.22, 0.16, 0.14 μm average diameter, respectively; M: Proteins marker; The arrows show the labeled proteins.

Fig. 3 Affect of elution on the combination of protein with rubber particle having different diameter

Fig. 4 Combination of different proteins with small rubber particles in vitro by incubation 1-6: The samples of B-serum, C-serum, 85% ammonium sulfate precipitation of B-serum, purified glucanase and 85% ammonium sulfate precipitation of C-serum, respectively; B6, as the control, showing the incubation of small rubber particle with the buffer. M: Proteins marker. The short arrow shows the high-abundant hevein protein in lane 3, the long arrow shows the condensed 37 kDa protein in lane 5, and the other arrows show the labeled proteins in western-blotting.

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