GPA5基因调控水稻贮藏蛋白后高尔基体分选的分子机理研究

Mutants with defects in storage protein sorting are excellent genetic resources for studies on protein quality improvement in rice. We previously identified three essential regulators involved in post-Golgi trafficking of storage proteins in rice, including GPA1/OsRab5a, GPA2/OsVPS9a and GPA3/OsKelch1. Combined cytological, molecular and biochemical studies revealed that GPA3 forms a regulatory complex with OsRab5a via its interaction with OsVPS9a. Furthermore, genetic data demonstrated that GPA3 acts synergistically with GPA1 and GPA2 to regulate DV (Dense vesicle)-mediated post-Golgi traffic in rice. Recently, using two allelic post-Golgi sorting defect mutants, we identified another novel regulator with a PX domain required for lipid binding. Using cytological, biochemical and molecular biological technology and methods, this project will focus on studies on the developmental defects of endomembrane system in gpa5 endosperm cells and the molecular mechanism of GPA5 gene in regulating storage protein sorting. The completion of this project will expand our understanding of vesicle-mediated storage protein trafficking pathways and provide gene resourses and theoretical directions for protein quality improvement in rice.

贮藏蛋白分选缺陷突变体是稻米蛋白品质改良研究的良好遗传材料。前期我们通过正向遗传学的研究鉴定了3个调控水稻贮藏蛋白后高尔基体分选的关键因子：GPA1/OsRab5a、GPA2/OsVPS9a和GPA3/OsKelch1，功能研究证实三者可形成一个功能蛋白复合体，协同调控贮藏蛋白的后高尔基体分选。近期，我们通过两个等位突变体分离了另外一个新的调控贮藏蛋白后高尔基体分选的关键因子GPA5，该蛋白含有一个可以特异结合磷酸肌醇-3-磷酸和磷酸肌醇-4-磷酸的结构域-PX。本项目拟进一步通过细胞、生化和分子生物学的技术手段，重点研究gpa5突变体胚乳发育的细胞学特性和GPA5基因的生物学功能，以期明确该基因调控水稻贮藏蛋白后高尔基体分选的分子作用机制，为全面解析水稻贮藏蛋白合成、分选和沉积的分子网络途径奠定基础，并为稻米蛋白品质改良提供遗传资源和理论支撑。

致密囊泡（DV）是植物种子细胞特有的一类运载工具，负责植物贮藏蛋白由高尔基体到蛋白贮藏液泡（PSV）的运输。然而，调控DV-PSV定向转运的分子机制尚不清楚。我们在前期工作中鉴定了一个水稻谷蛋白定向转运缺陷的突变体gpa5。利用半薄切片和超薄切片观察显示，gpa5中DV发生错误分选，导致大量谷蛋白被转运到胞外体空间。图位克隆证实GPA5编码一个植物特有的蛋白质，是拟南芥EREX的同源蛋白。生化实验GPA5是一个膜相关的蛋白质，可以形成同源二聚体。亚细胞定位显示GPA5在胚乳细胞中定位在DV外围。共定位、生化和遗传证据表明GPA5与我们前期鉴定的Rab5a和VPS9a在同一通路中负责DV介导的谷蛋白运输，GPA5作为Rab5a的效应因子发挥功能。我们进一步证实GPA5可以招募下游的栓系复合体CORVET和融合复合体SNARE，介导DV与PSV的融合。通过上述实验，我们证实GPA5充当了Rab5a的效应子，介导DV与PSV的栓系和膜融合，这些研究拓展了我们对植物特有的囊泡转运系统的认知，也为稻米蛋白品质改良提供了基因资源和材料基础。