转录因子OsLFT1与OsLIP1互作调控水稻开花期的分子机制

水稻开花期调控是多基因参与的复杂生命过程。申请者前期研究发现，水稻bHLH转录因子家族基因OsLFT1的突变体oslft1s开花期较野生型提前，而超表达OsLFT1导致水稻开花期显著延迟，但其分子机制尚不清楚。本项目拟利用oslft1s突变体和OsLFT1超表达转基因株系为试材，利用染色质免疫共沉淀等技术，筛选OsLFT1调控开花期的靶基因，并通过CRISPR/Cas9技术创制靶基因的突变体、解析其功能。利用凝胶阻滞分析和双荧光素酶报告系统等方法，分析OsLFT1的DNA结合能力和激活活性。利用酵母双杂交和免疫共沉淀等技术，筛选OsLFT1的互作蛋白 (OsLIP1)，确定介导它们互作的结构域，并通过CRISPR/Cas9技术创制OsLIP1的突变体，解析OsLIP1的功能。研究结果可以揭示OsLFT1和OsLIP1互作调控水稻开花期的分子机制，为水稻开花期的遗传改良奠定基础。

The regulatory mechanisms on flowering time control are complicated which are involved in multiple genes. The results of our previous studies show that mutants of OsLFT1, a member of bHLH transcriptional factor family, termed oslft1s, exhibits early flower phenotype; overexpression of OsLFT1 delays flowering time in rice; however, the molecular mechanisms remain obscure. To further explore the roles of OsLFT1 in regulating flowering time of rice, the oslft1s early flowering mutants and late flowering OsLFT1 overexpression transgenic lines will be applied. The target gene through which OsLFT1 regulates flowering time will be identified by using ChIP-sequencing and RNA-high throughput sequencing techniques. By using CRISPR/Cas9, mutants of the target gene will be generated to elucidate its functions. Furthermore, the DNA binding and activation activity of OsLFT1 will be analyzed by ChIP-qPCR, Gel shift assay, Dual luciferase reporter assay, and Yeast Two Hybrid. To identify the partner of OsLFT1, termed OsLIP1, OsLFT1 will be used as bait to screen the cDNA library of rice through Yeast Two Hybrid. CO-IP and SFLC assay will be applied to confirm the interaction. Mutants of OsLIP1 will be generated to dissect its functions by using CRISPR/Cas9 approach. The findings of the study will broad our understanding of the roles of OsLFT1 associated with OsLIP1 in flowering time control of rice and provide sound theoretical and practical foundation to improve rice variety and increase the adaptability, yield, and quality of rice.