水稻转录因子OsCOL5调控光周期开花的分子机制研究

Flowering time (heading date) is an important ecological trait that determines growing seasons and regional adaptability of plants to specific natural environments. Recent studies show that members of the CO-like gene family have been shown to play important roles in the photoperiodic regulation of flowering in many plant species and are conserved between monocots and dicots, over-expression of CONSTANS-like 5 can induce flowering in short-day grown Arabidopsis. While the ortholog of Arabidopsis COL5 in rice have not been reported. In our previous study, we found over-expression of OsCOL5 can suppress flowering under long-day conditions, while the molecular mechanism of controlling flowering is unclear. Based on previous work, in this project, we will clarify the location of OsCOL5 in the photoperiodic flowering pathways in rice by using qRT-PCR combined with genetic experiments; screening the interacting protein of OsCOL5 by yeast two-hybrid assay and the binding promoter sequences of downstream genes of OsCOL5 by bacterial one-hybrid; also carrying out domestication analysis of OsCOL5 to study the genetic diversity, variation pattern and selection model, ultimately exploring the molecular mechanisms of OsCOL5 which controlling flowering in rice. The implementation of this project will also provide new ideas and insights into the mechanisms for COL gene family in rice flowering regulation.

开花时间（抽穗期）是决定水稻品种的生长季节和地区适应性从而适应特异自然环境的重要农艺性状。研究发现COL基因家族成员广泛参与高等植物光周期开花调控，并且在单子叶和双子叶植物之间高度保守，过表达拟南芥COL5基因在短日照条件下促进开花。然而，COL5在水稻中同源基因的功能一直未见报道。申请人在前期的研究工作中发现过表达OsCOL5基因在长日照条件下抑制开花，然而其调控开花的分子机制尚不清楚。本项目拟在已有工作基础上利用qRT-PCR结合遗传实验阐明OsCOL5基因在水稻光周期开花调控途径中的位置，通过酵母双杂交和细菌单杂交筛选OsCOL5蛋白的互作蛋白和OsCOL5基因结合的下游基因启动子序列，并对OsCOL5基因进行驯化分析，研究其遗传多样性、变异规律和受选择状况，最终探索OsCOL5基因调控水稻开花的分子机制。本项目的实施还将为认识COL基因家族参与水稻开花调控的机制提供新的思路和见解。

抽穗期是水稻最重要的农艺性状之一，决定品种的地区和季节适应性。成花转变是水稻生长发育中的一个关键节点，因此深入研究水稻成花转变的机理有重要意义。本项目通过在水稻品种日本晴中过表达OsCOL5基因，发现转基因植株在杭州自然长日照和海南自然短日照条件下均呈现明显的晚抽穗表型。通过亚细胞定位实验发现OsCOL5蛋白定位在细胞核中。转录自激活实验表明OsCOL5具有很强的转录自激活活性，对OsCOL5基因的缺失分析显示位于BBX结构域和CCT结构域之间的区域对转录活性是必须的。通过组织表达模式分析发现OsCOL5在幼嫩叶片中表达量较高，节律表达模式分析发现OsCOL5呈现明显的昼夜节律表达。RT-qPCR实验发现OsCOL5位于Ghd7上游，通过促进Ghd7表达，抑制Ehd1、Hd3a、RFT1、OsMADS14和OsMADS15表达最终抑制抽穗。该结果为全面揭示OsCOL5基因调控水稻光周期开花的分子机制奠定了基础。此外我们还研究了OsCOL16和OsCOL15基因控制水稻抽穗的分子机理。过表达OsCOL16在长日照和短日照条件下均抑制开花。进一步研究发现OsCOL16通过上调Ghd7来抑制Ehd1、Hd3a和RFT1的表达，最终延迟抽穗。遗传多样性和进化分析表明OsCOL16的两个等位基因的抽穗期具有显著差异，分子生物学和亲缘地理学分析表明OsCOL16在调节水稻光周期开花过程中起重要作用。OsCOL15定位在细胞核中并具有转录自激活活性，并且OsCOL15基因具有节律表达模式。通过遗传转化实验，证明过表达OsCOL15基因在短日照和长日照条件下均延迟水稻抽穗。其在水稻光周期开花途径中位于Ghd7和RID1的上游。这些研究成果为深入解析CONSTANS-like基因调控水稻光周期开花的分子机理奠定了很好的基础。