运用BioID技术筛选水稻GS3互作蛋白

Screening of interaction proteins of GS3 in rice using BioID

关键词:  BioID, GS3, 水稻, 籽粒大小, 互作蛋白

Key words:  BioID, GS3, rice, grain size, interaction proteins

摘要:

水稻异源三聚体G蛋白系统中的非典型γ亚基GS3,是一个控制籽粒大小的主效数量效应基因座,在调节籽粒大小中发挥负调控因子的功能。BioID(proximity-dependent biotin identification)为邻近蛋白标记技术,其工作原理是生物素连接酶能使其周围的蛋白带上生物素,同时生物素又能和链霉亲和素紧密结合,所以能够利用链霉亲和素偶联的磁珠富集目标蛋白。该技术具有灵敏、高效和周期短等特点,为筛选互作蛋白提供了新方法。为了解析GS3的蛋白调控网络,该研究以水稻原生质体为材料,采用BioID 技术对GS3在水稻中的互作蛋白进行了筛选。Western-blot结果表明:融合蛋白BirAG-GS3在原生质体中成功表达并生物素化GS3邻近蛋白。使用链霉亲和素磁珠富集生物素化后的蛋白,并进行蛋白质谱测序,获得了与GS3邻近的可能存在直接或间接互作的蛋白。将获得的蛋白进行功能富集与注释,并构建蛋白-蛋白互作网络。对部分蛋白进行了BiFC 验证,发现GS3可能与ICL、PPDK、RPN7和RH15发生相互作用,涉及能量代谢的调节、种子淀粉物质的储存、泛素-蛋白酶体系统以及凋亡途径等生物过程。

Abstract:

GS3, encoding the noncanonical γ subunit of heterotrimeric G proteins system in rice, is a major quantitative trait locus for grain size in different rice varieties, serving as a negative regulator in seed or organ size. Newly-developed BioID(proximity-dependent biotin identification)system, which detects proximal proteins in vivo, has been successfully applied in different species for its features of sensitivity, high efficiency and fast speed. And the mechanism of BioID is that the biotin ligase, the core component, could attach biotin to the proximal proteins, and the biotin also connects tightly with streptavidin, therefore, the beads coupled streptavidin could be utilized for enriching the target proteins. In this study, we screened proteins interact directly or indirectly with GS3 in rice protoplasts by BioID method. The results of Western-blot showed that fused protein BirAG-GS3 expressed and biotinylated the proximal proteins of GS3 in the protoplasts successfully. Biotinylated proteins were enriched by beads coupled streptavidin and the Mass Spectrometry sequencing performed on it. Then, the function of proteins has been annotated and enriched, meanwhile, protein-protein interaction graph was established. Some of the candidate proteins were confirmed by BiFC, indicating the possible interaction between ICL, PPDK, RPN7 and RH15, and the engagement of GS3 in regulation of energy metabolism, stock of starch, ubiquitin-proteasome system and apoptotic pathway. The above results will facilitate the elucidation of the regulation network of the function of GS3.