水稻 OVATE Family Protein 8 （OsOFP8）基因的功能研究

OVATE gene was first identified as a key regulator of fruit shape in tomato, OVATE family proteins (OFPs) are characterized as plant-specific transcription factors. Rice genome contains 31 OFPs, the roles of these OsOFPs involved in plant development are not understood. Brassinosteroids (BRs) are a class of steroid hormones involved in diverse biological functions. OsGKS2 play a critical role in BR signaling by phosphorylating downstream components such as OsBZR1 and DLT. We found that OsOFP8 plays a positive role in BR signaling pathway. The gain-of-function mutant Osofp8 and OsOFP8 overexpression lines show enhance lamina joint inclination and altered grain shape, whereas OsOFP8 RNAi transgenic lines are less sensitive to BL treatment. Further analyses indicate that OsOFP8 is able to bind to the promoter region of OsBZR1 to regulate its expression. Furthermore, OsOFP8 interacts with and is phosphorylated by OsGSK2, phosphorylated OsOFP8 shuttles to the cytoplasm and is targeted for the proteasomal degradation. These results demonstrate that OsOFP8 is a substrate of OsGSK2. To study the mechanisms of OsOFP8 by which it regulates lamina angle and grain shape, we are about to generate OsOFP8-YFP transgenic rice plants to monitor OsOFP8 levels during the treatments of BL and BRZ, also we are going to carry out ChIP-Seq and RNA-Seq analysis to identify potential gene targets of OsOFP8, as well as to perform the yeast two hybrid screening to find the OsOFP8-interacted proteins. To this end, we can explore the functions of OsOFP8 at molecular level in rice.

水稻叶夹角大小及籽粒形状是水稻生产上的两个重要性状，但这两性状的分子调控网络并不清楚。OFP蛋白是一类控制果实/种子形状的转录调控因子，目前几乎没有关于水稻OFP基因功能的报道。OsOFP8影响水稻叶片夹角及籽粒形状，并参与油菜素内酯（BR）信号传导；OsOFP8定位于细胞核内并能结合到DNA序列上；OsOFP8能与OsGSK2互作并被磷酸化，磷酸化的OsOFP8能穿梭到细胞质中被降解，但其作用机制不清楚。本项目将详细研究OsOFP8在BR信号传导途径中的作用及OsOFP8的分子调控网路，从而解释OsOFP8对叶夹角及籽粒大小的调控机制。通过获得OsOFP8-YFP转基因水稻，详细研究OsOFP8在BR途径中的作用；利用ChIP-Seq和RNA-Seq技术研究OsOFP8调控的靶基因；通过酵母双杂交系统，研究OsOFP8的互作蛋白。通过以上系列分析，明确OsOFP8的分子调控机制。

水稻叶夹角大小及籽粒形状是水稻生产上的两个重要性状，研究这两个性状的基因调控网络对水稻生产上调控这两个性状，提高水稻产量有重要的作用。OFP8在水稻叶夹角大小及籽粒形状的形成中起重要作用，OFP8 参与油菜素内酯（BR)的信号传导途径，和BR途径中的重要负调控蛋白GSK2相互作用，并被GSK2 磷酸化，磷酸化后的OFP8 会从细胞核中转移到细胞质中，并通过蛋白酶途径被降解。OFP8还能够和14-3-3蛋白发生相互作用，这种相互作用能使OFP8蛋白滞留在细胞质中，影响OFP8在细胞核内的含量，从而影响OFP8在核内的转录调控功能。OFP8还能和同家族内的OFP19互作，而过表达OFP19的转基因水稻和过表达OFP8的转基因水稻杂交的F1代植株的表现观察显示OFP8对叶夹角和籽粒形状的调控作用被OFP19所抑制，表明在OFP8和OFP19的互作中，OFP19对OFP8表现为上位效应。 同时，通过酵母双杂交实验发现，OFP8和F-box蛋白互作，和F-box的互作可能是导致OFP8降解的原因。 RNA-Seq技术发现OFP8 能调控许多代谢途径和激素信号传导途径相关的基因的差异表达。这些研究结果对深入理解OFP基因的功能和水稻叶夹角及籽粒形状的形成有重要帮助。