OsHAP15调控水稻开花的分子机制研究

Several HAP genes regulate flowering in rice. In order to understand how many members in the HAP family involve to regulating flowering, we produced overexpression mutants and knockout mutants for all HAP members by overexpression technique and CRISPR-Cas9 system. Of them, the knockout mutant of OsHAP15, one member of HAP3 subfamily, delayed flowering under long day conditions and promotes flowering under short day conditions. The functional complementation of OsHAP15 rescued the phenotype of OsHAP15 mutant. Therefore OsHAP15 is a new flowering gene. On the basis of the results mentioned above, we will uncover the molecular mechanism of OsHAP15 regulating flowering in this project. We first characterize OsHAP15 by examining its spatial and temporal expression pattern and subcellular localization of its encoding protein. We will identify its downstream genes by ChIP-Seq assay and qRT-PCR assay; and identify the target genes, which are directly bound by OsHAP15 via EMSA and yeast one-hybrid assay. We will search for the proteins interacted with OsHAP15 by screening a leaf-tissue yeast two-hybrid cDNA library, testing interaction using yeast two-hybrid system, BiFC and Pull-Down techniques, especially for the proteins of HAP2, HAP5 and CCT families interacted with OsHAP15. Yeast three-hybrid assay will be used to validate the trimeric complex OsHAP15 involved. Finally we will construct regulatory network OsHAP15 mediated and enrich rice flowering regulatory mechanism.

多个HAP家族基因参与水稻开花调控。为了明晰有多少HAP家族成员参与调控开花，我们利用超表达和CRISPR-Cas9技术得到HAP家族所有成员的超表达和功能敲除突变体，其中HAP3亚家族成员OsHAP15敲除突变体长日条件下提前7天开花，短日条件下延迟10天开花，OsHAP15互补转化使敲除突变体表型回复，因此OsHAP15是一个开花基因。我们在此基础上，开展OsHAP15调控开花机制研究。通过检测OsHAP15时空表达特征和亚细胞定位明确它的自身特点。利用ChIP-Seq和RNA-Seq分析寻找其下游靶基因；利用酵母单杂交和EMSA分析鉴定与OsHAP15直接结合靶基因。通过酵母双杂交文库筛选、酵母双杂交实验、BiFC和Pull-Down技术寻找其互作蛋白；利用酵母三杂交系统验证可能存在的HAP家族三聚体成员，建立OsHAP15介导的开花调控网络，丰富水稻开花调控机制。

抽穗期是关系到作物产量的重要的农艺性状之一。多个HAP家族基因参与水稻开花调控途径。前期通过超表达和CRISPR-Cas9技术得到HAP家族所有成员的超表达和功能敲除突变体，其中HAP5亚家族成员OsHAP15敲除突变体长日条件下延迟5天开花，短日条件下延迟3天开花，OsHAP15互补转化使敲除突变体表型回复，因此OsHAP15是一个开花基因。本项目希望通过OsHAP15功能解析，丰富水稻调控开花的网络。OsHAP15是定位在细胞核内但无转录活性的转录因子，它主要在叶片中表达。结合RNA-seq和RT-PCR发现OsHAP15-GFP超表达植株中，Ehd1、Hd3a、RFT1等开花促进因子的表达量被抑制，但是Hd1、Ghd7、Ghd8等基因并未出现显著差异。说明OsHAP15同Ghd8一样，是通过Ehd1途径进行水稻抽穗期的调控。研究发现，OsHAP15蛋白体内体外都与开花相关蛋白Ghd8, Ghd7, Hd1互作，而Ghd8蛋白体内体外都与Ghd7或者 Hd1互作。然而OsHAP15，Ghd8无转录活性，且OsHAP15与Hd1的互作抑制Hd1的激活活性; Hd1可以结合到HAP15下游调控基因SUI的启动子上。因此推测，OsHAP15可能通过与Ghd8及Hd1互作形成复合物，同时Hd1行使转录功能，调控水稻开花基因。