橡胶粒子洗脱液中SRPP-HRBP-REF膜蛋白复合体成员及其互作蛋白的分离鉴定和功能分析

Natural rubber is an important raw material for national defense industry. Rubber particle (RP) is a specific organelle for natural rubber biosynthesis and storage. The biosynthesis of high molecular weight rubber molecules was performed by using membrane protein complex localized at RP membrane. These rubber particle membrane proteins may be loosely combined with membrane, or closely combined with membrane to formation protein complexes. In the past years, to identify the membrane proteins, total rubber particles are collected in several washes with different solutions. Then, membrane proteins are isolated from the washed rubber particles. However, our primary proteomic analysis of the discarded washing solutions (WS) revealed that a large amount of proteins, including Nogo-B receptor named HRT1-REF bridging protein (HRBP), rubber elongation factor (REF) and small rubber particle protein (SRPP), were identified from the washed solutions. We think that these washing solution proteins are likely to loosely combine with membrane to form protein complex and participate in rubber synthesis process of rubber particles. These proteins and their interaction molecules, probably in small rubber particles and washed solutions, can form a protein complex for rubber biosynthesis and transport. There is a kind of protein machinery on small rubber particles, and this protein machinery might be activated through posttranslational modifications of protein isforms to operate the precise functions of the machinery. This project aims to identify protein complex and the proteins which had an interaction with REF, HRBP and SRPP from rubber particles and their washed solutions. Then, screened important proteins to verify their function by in vitro synthesis system of latex, and selected part of the crucial proteins for functional analysis. The results will have tended to analysis the protein composition of rubber particle membrane protein complex and their function, will contribute to further understanding protein chemical mechanism of rubber biosynthesis and its regulation mechanisms, and also provide new theoretical foundation for the regulation of natural rubber biosynthesis.

天然橡胶是重要的国防战略物资，橡胶粒子是天然橡胶合成和贮存的特殊细胞器，天然橡胶通过其膜蛋白复合体合成橡胶烃。过去一直通过反复洗脱来纯化橡胶粒子，导致很多膜结合蛋白丢失。我们新设计了一种橡胶粒子洗脱液蛋白提取方法，进行洗脱液蛋白质组初步研究，发现洗脱液中含有小橡胶粒子膜蛋白SRPP，橡胶延伸因子REF和橡胶桥联蛋白HRBP等蛋白酶。我们推测，洗脱液中很可能含有SRPP-HRBP-EF膜蛋白复合体的主要成员，一些成员及其互作蛋白可能与橡胶粒子膜非紧密结合，形成复杂的蛋白质机器，通过蛋白修饰来精确调控橡胶生物合成。本项目拟用蛋白质化学和分子生物学等技术，从橡胶粒子及其洗脱液中鉴定更多膜蛋白复合体成员及互作蛋白，并鉴定经乙烯刺激后蛋白修饰规律，进行部分关键蛋白体外功能模拟验证。结果有望解析橡胶粒子及其洗脱液中膜蛋白复合体的具体构成和作用方式，为阐明天然橡胶合成的蛋白质调控机制提供新的理论依据。

天然橡胶是重要的国防战略物资，橡胶粒子是天然橡胶合成和贮存的特殊细胞器，天然橡胶通过其膜蛋白复合体合成橡胶烃。过去一直通过反复洗脱来纯化橡胶粒子，导致很多膜结合蛋白丢失。我们利用自己团队新设计的橡胶粒子洗脱液蛋白提取方法，进行洗脱液蛋白质组研究，鉴定了2930多个蛋白，包括小橡胶粒子膜蛋白SRPP，橡胶延伸因子REF和橡胶桥联蛋白HRBP等。我们进一步对分离纯化的大橡胶粒子和小橡胶粒子进行蛋白质组全谱鉴定，分别鉴定了1500和1990个左右的蛋白质，这些蛋白质中，同样包含有上述蛋白。因此，我们发现，橡胶粒子及其洗脱液中确实含有SRPP-HRBP-REF膜蛋白复合体的主要成员，一些成员及其互作蛋白可能与橡胶粒子膜非紧密结合，形成一个复杂的蛋白质机器。对乙烯刺激前后胶乳及橡胶粒子进行蛋白质组研究发现，该蛋白质及其主要通过磷酸化蛋白修饰来精确调控天然橡胶生物合成。我们进一步通过蛋白互作等技术，鉴定了更多与SRPP、HRBP或REF有互作关系的蛋白，并将其中的部分关键蛋白体进行橡胶草遗传转化实验，获得了含胶量显著提高的转基因橡胶草植株，为进一步开发利用转基因橡胶草和橡胶树提供了前期基础，为阐明天然橡胶合成的蛋白质调控机制提供了新的理论依据。