适于混播制种的水稻红颖基因RH4调控类黄酮生物合成的机理

The discovery and utilization of specific genetic resources provided a new strategy for innovative seed production technology. The rice mutant red hull 4 (rh4) which exhibits high total flavonoids and anthocyanin contents has been identified in our previous work. Compared with other marker traits, the stable red hull phenotype of rh4 mutant is more powerful and intuitive for rapid selection of hybrid seeds to solve the current critical technical problems in mixed-sowing seed production. Recently, candidate gene RH4 was obtained through fine mapping approach and sequence analysis. RH4 encodes an leucine-rich repeat receptor-like serine/threonine-protein kinase FLAGELLIN SENSING 2 (LRR-RLK-FLS2). The mutation of RH4 caused that the relative expression level of some key genes related to the flavonoid biosynthesis were changed significantly. However, at present, it is unclear about the biological function of RH4 and its specific mechanism of action. Therefore, based on the previous research, we intend to study RH4 biological function, identify its interacting protein，clarify its acting site and control pattern in flavonoid biosynthesis pathway and reveal the molecular regulation mechanism It will provide new genetic resources and theoretical support for the utilization of mixed-sowing seed production in the hybrid rice.

特异遗传资源的发掘与利用为制种技术创新提供了新途径。red hull 4(rh4)是申请人前期获得的隐性红颖水稻突变体，其颖壳总黄酮和花青素含量显著高于野生型。利用rh4稳定红颖特征作为分选标记，可解决当前混播制种策略的关键技术难题，实现快速直观的杂交种精准分离。申请人已通过精细定位和测序分析获得候选基因RH4，其编码富亮氨酸重复片段类受体丝氨酸/苏氨酸蛋白激酶（LRR-RLK-FLS2）。RH4突变后导致多个参与类黄酮生物合成的关键基因发生显著表达差异。目前并没有LRR-RLK参与类黄酮生物合成途径的相关报道，RH4的生物学功能也不清楚。本项目拟在此基础上，研究RH4的生物学功能，鉴定其互作蛋白，明确其参与类黄酮生物合成的作用位点及调控方式，揭示RH4调控类黄酮生物合成的分子机理，为适应于混播制种的杂交稻亲本定向遗传改良提供新的基因资源和理论支撑。

加强水稻红颖基因的鉴定和利用，对于解析类黄酮生物合成的遗传调控机制，建立杂交稻混播制种技术具有重要意义。本项目围绕水稻红颖突变体rh4的图位克隆与功能开展研究，取得以下结果：（1）rh4颖壳显著积累类黄酮，受单个隐性细胞核基因控制；利用图位克隆策略将rh4精细定位于12号染色体，遗传互补试验证实编码一段信号肽和一个跨膜结构域的LOC_Os12g10880为rh4的目的基因。（2）转录组学、蛋白组学和代谢组学综合分析发现，RH4参与类黄酮、苯丙氨酸等的生物合成，rh4中大波斯菊甙等黄酮醇类含量的上调是形成红颖性状的生理成因。（3）RH4蛋白定位于细胞核与部分细胞膜，在根、叶和颖壳等部位中表达；运用酵母双杂交、双分子荧光互补等技术，验证了RH4与OsDSK2a、OsDSK2a与OsCHS存在互作关系，提出了RH4介导OsDSK2a与CHS的相互作用来调控类黄酮的生物合成模型。（4）大田试验发现“红颖/非红颖”组合的混播制种产量显著高于传统制种，表明rh4是适于杂交稻混播制种的理想遗传工具。这套技术通过了现场评议。申请专利3项，发表论文9篇。培养博士4名，培养硕士4名。