梨生物钟转录因子LCL1调控成花的分子机制

Circadian clock is involved in numerous gene transcription regulation process, play crucial roles in growth and development process, make plants able to forecast the internal and external changes. So that the clock can enhance the adaptability of plants. Our preliminary data suggest the expression of flowering related genes have significant rhythms phenomenon in pear, indicating that it is regulated by the pear clock system, but its molecular mechanism is unclear. LCL1 is an potential key component of pear clock system, belonging to the MYB transcription factors family. This project intends to characterize the feather of pear LCL1 from the following four aspects: analyzing LCL1 protein complex by yeast-two-hybrid screening and bimolecular fluorescence complementation analysis; detecting binding of LCL1 protein to the target DNA by yeast-one-hybrid and electrophoretic mobility shift assay; analyzing transcriptional activity by transient expression in protoplast and dual-luciferase reporter assay system; then verification the gene function by transgenic plant analysis. Above all, this project sought to elucidate the function of pear clock component LCL1 and its role in flowering regulation. These studies will help us further understanding of the signal transduction in pear development and promote pear breeding process.

生物钟是植物内源性计时机制，参与调控诸多生长发育过程，使植物能够积极响应内外环境变化。梨树的成花是其生长发育的关键步骤和形成果实的前提条件，申请人前期实验发现梨成花相关基因的转录水平具有显著的生物节律现象，这表明其受生物钟系统调控，但其分子机制尚不清楚。LHY-CCA1 LIKE 1（LCL1）是梨生物钟重要的上游组分，属于MYB转录因子。本项目拟从以下四个方面探究生物钟转录因子LCL1对梨成花过程的调控机制：采用酵母双杂交筛选LCL1互作蛋白，并用双分子荧光互补方法验证；采用酵母单杂交和凝胶阻滞等方法分析LCL1及其互作蛋白对成花基因启动子的结合能力；采用原生质体瞬时表达体系分析LCL1对梨成花基因的转录调节活性；并通过转基因材料做进一步的功能验证。本研究力图阐明生物钟组分LCL1调控梨成花的分子机制，完善梨生长发育调控的科学理论，也将为调控梨开花、加快梨育种进程提供基因资源。

梨树的成花是其生长发育的关键步骤和形成果实的前提条件，本项目通过基因组分析、克隆和功能验证找到了具有调节开花作用的4个转录因子PbLCL1，PbLCL4，PbCOL8和PbSPL15。梨PbLCL1，PbLCL4是两个MYB类转录因子，两者都受到生物钟和光周期的调控。 PbLCL1-GFP和PbLCL4-GFP均定位于细胞核且可以相互作用。PbCOL8位于光周期信号下游，具有显著的开花抑制活性。PbSPL15与年龄相关，过表达PbSPL15可以促进开花进程。本研究通过实验验证，找到了梨中新的开花调控基因，并对这些基因间的调控关系进行了探索，为调控梨童期和开花时间提供了基因资源。