Resolution of accumulation mechanism into protein body of prolamin in rice seed

水稻中富含半胱氨酸的醇溶谷蛋白由10 kDa、15 kDa和16 kDa三种多肽组成，与13 kDa的贫半胱氨酸醇溶谷蛋白形成醇溶谷蛋白蛋白体。免疫荧光和免疫电子显微镜表明，10和13 kDa的醇溶谷蛋白定位于中央和外周区域的PB。此外，免疫荧光显微镜显示，10 kDa醇溶谷蛋白积累到内质网腔在开花后7天，之后，13 kDa醇溶谷蛋白的10 kDa醇溶谷蛋白的核心层。在13 kDa半胱氨酸贫乏的醇溶谷蛋白缺陷突变体esp1中，观察到球形PB，其电子致密核心包括10 kDa醇溶谷蛋白。在10 kDa醇溶蛋白缺陷突变体esp3中，未观察到PB的核心结构，而是观察到不规则的、电子透明的和肥大的PB。这些结果表明，在水稻醇溶谷蛋白PB的形成开始积累的10 kDa醇溶谷蛋白在中央核心和顺序的13 kDa醇溶谷蛋白是分层的核心结构。结论：10 kDa醇溶蛋白在水稻中起着形成核心结构以稳定醇溶蛋白PB结构的作用。Esp1基因的鉴定和功能在esp1突变中，半胱氨酸缺失的醇溶蛋白减少。为了明确esp1突变的致病基因，构建了esp1基因的高密度连锁图谱，并进行了互补试验。结果表明，Esp1基因编码翻译终止因子真核释放因子1（eRFF1），表明eRF1是调节半胱氨酸贫乏醇溶蛋白翻译的因子。

Accumulation of prolamin into protein bodyIn rice cystein rich prolamin, which is composed of three polypeptides, 10 kDa, 15 kDa, and 16 kDa, forms the prolamin protein body with 13 kDa cystein poor prolamin. Immunofluorescent and immunoelectron microscopies indicated that 10 and 13 kDa prolamins localized to central and peripheral regions of the PB. Additionally, immunofluorescent microscopy showed that 10 kDa prolamin was accumulated into ER lumen at 7 day after flowering and after that 13 kDa prolamin was layered over the core of 10 kDa prolamin. In the 13 kDa cystein poor prolamin deficient mutant, esp1, spherical PB with the electron dense core including 10kDa prolamin was observed. In the 10 kDa prolamin deficient mutant, esp3, the core structure of PB was not observed, instead, irregular, electron lucent and hypertrophied PB was observed. These results suggest that the formation of prolamin PB in rice is initiated by accumulation of 10 kDa prolamin in the central core and sequentially 13 kDa prolamin is layered over the core structure. It is concluded that 10 kDa prolamin plays a role to form the core structure to stabilize prolamin PB structure in rice.Identification and function of Esp1 geneIn esp1 mutation, cystein poor prolamin is reduced. In order to specify the responsible gene of esp1 mutation, the construction of the high-density linkage map of esp1 gene and the complementation test were performed. As results, it became clear that Esp1 gene encode the Eucaryotic Release Factor 1(eRFF1), which is translation termination factor, indicating that the eRF1 is a factor regulating the translation of cystein poor prolamin.