一个新的水稻BR信号调控因子DLB的鉴定和功能研究

Rice architecture closely relate to the yield, and Brassinosteroid (BR) is one of most important plant hormone that regulating architecture. It has great theoretical and practical value to investigate and take full advantage of plant BR signaling to improve rice yield. However, compared with advance of BR signal in Arabidopsis, less is known in Rice.. In previous study, we characterized a rice material dlb-D in which Dlb（Dwarf and leaf bending）is over expressed. Interestingly, dlb-D shows multiple enhanced BR signal phenotype, including decreased plant height, increased leaf angle and larger seed size. Through analysis of dlb-D and dlb-k in which the Dlb gene is knocked via CAS9 mediated genome editing technique, we found that DLB positively regulated the response of leaf angle to BR treatment; expression of some BR biosynthesis genes (D2, DW) is regulated by Dlb in an negative feedback manner; expression of BR-induced genes (OsXTR1, OsBU1) is up-regulated in dlb-D and down-regulated in dlb-k. All these data imply that DLB is a novel positive regulator in rice BR signal. DLB is a transcriptional factor and localized in nucleus. Further investigation show that DLB can inhibit expression of OsGSK2 possiblly by direct binding the conserve motif in OsGSK2 promoter. However, the molecular mechanism of DLB is not clear thus far. In this proposal, first, we will construct and analyze the phenotype of double mutant between the dlb and known BR signal components to prove the DLB is involved in rice BR signal. Second, we will investigate the mutual regulation relationship between the DLB and known BR signal components, especially OsGSK2, by performing multiple techniques. Finally, we will reveal the molecular mechanism of Dlb functioned in rice BR signal.

水稻株型与产量密切相关，油菜素内酯（BR）是调控株型的主要激素之一。研究和利用BR信号途径来提高水稻产量，具有重要的理论和应用价值。在前期构建的水稻转录因子过量表达的突变体库中，鉴定到一个呈现典型BR过量表型的材料，表现为植株变矮、叶夹角变大、种子变大，命名为dlb-D。对Dlb基因过表达和敲除的水稻研究发现，Dlb基因正调控叶角对BR的反应；BR诱导表达基因OsXTR1 和OsBU1等受Dlb正调控；BR合成基因D2和DW等的表达受Dlb负反馈抑制，表明 DLB可能是水稻BR信号一个新的正调控因子。更有意思的是，DLB有可能直接抑制BR途径负调节因子OsGSK2的表达。本申请拟从两方面入手，一是分析DLB与已知BR信号材料的双突变体的表型，在遗传学上证明DLB参与BR信号；二是解析DLB与BR信号组分、特别是与OsGSK2的相互调控关系。最终揭示DLB参与水稻BR信号途径的分子机理。

通过本项目的资助。我们筛选水稻转录因子过量表达的突变体库，筛选获得Dlb-D（Dwarf and leaf bending）突变体，经后期验证发现Dlb基因即为转录因子OsWRKY53基因。OsWRKY53过表达转基因水稻（OsWRKY53-OE）呈现出叶倾角增大、粒型增大、其叶倾角对外源的BR处理超敏感等一系列BR信号增强的表型；而敲除突变体（oswrky53）表现出叶倾角变小、粒型变小、株高变矮、其叶倾角对外源的BR处理敏感性下降等一系列BR信号缺陷的表型，说明OsWRKY53是BR信号的一个新的正调控因子。进一步研究分析发现，OsWRKY53与丝裂原激活蛋白激酶OsMAPK6之间存在互作关系，并且OsMAPKK4-OsMAPK6能够磷酸化OsWRKY53提高后者的生物活性。此外，BR能够促进OsWRKY53蛋白水平的积累，抑制其转录水平的表达。以上结果表明，OsWRKY53是BR信号途径的一个新的正调控因子。. 同时，通过遗传学、生物化学、分子生物学等技术手段初步证实OsWRKY53作用于OsGSK2的遗传学下游，OsGSK2能够结合并磷酸化OsWRKY53。而且，OsWRKY53与OsBZR1协同调控BR生物合成基因的表达。. 目前，在该项目为第一标注，在国际植物学期刊 Plant physiology、Journal of Integrative Plant Biology上各发表学术论文1篇，授权专利1项，在投文章2篇。