橡胶树不同粒径橡胶粒子结合凝集相关蛋白的分析  

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

作　　者：丁欢 吴绍华[2] 夏志辉[1] 黄惜[1] 史敏晶 DING Huan;WU Shaohua;XIA Zhihui;HUANG Xi;SHI Minjing(College of Tropical Crops,Hainan University,Haikou,Hainan 570228,China;Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture andRural Affairs/State Key Laboratory Incubation Base for Cultivation and Physiology of Tropical Crops,Haikou,Hainan 571101,China)

机构地区：[1]海南大学热带作物学院,海南海口570228 [2]中国热带农业科学院橡胶研究所/农业农村部橡胶树生物学与遗传资源利用重点实验室/省部共建国家重点实验室培育基地-海南省热带作物栽培生理学重点实验室,海南海口571101

出　　处：《热带作物学报》2022年第12期2413-2421,共9页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31870590);海南省基金创新团队项目(No.320CXTD442);国家重点研发计划项目(No.2018YFD1000502)。

摘　　要：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。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 TricineSDS-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,andβ-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 p

关 键 词：橡胶树 橡胶粒子 凝集相关蛋白 蛋白免疫印迹 

分 类 号：S794.1[农业科学—林木遗传育种]