菊花独脚金内酯信号路径相关基因TOPLESS（CmTPL）调控开花的分子机理研究

Chrysanthemum has high ornamental and economic value, and plays an important role in the floriculture industry across the world. The high energy cost for the year-round products challenges the sustainable chrysanthemum producing. We found overexpressing the strigolactone(SL) biosynthesis gene CmCCD8 in chrysanthemum induced flowering of the transgenic plants comparing with the function of SL which delayed flowering in Arabidopsis, then the expression level of CmTPL whose homolog in Arabidopsis and rice plays a key role in the SL signaling was found lower in the transgenic chrysanthemum by RNA-seq, furthermore the transcripts of some flowering related key genes such as SOC1, AP1 and FUL were elevated in the plants; overexpression of CmTPL in Arabidopsis delayed flowering, while the mechanism involved in regulalting flowering time remained unclear. In present proposal, we aim to uncover the molecular mechanism of CmTPL in regulating the flowering time of chrysanthemum based on its interaction with CmJ using yeast two-hybrid screening. The bimolecular fluorescence complementation (BiFC), and GST Pull-down and Co-Immunoprecipitation（CoIP）will be used to confirm the interaction in vitro and in vivo. The function of CmTPL and CmJ will be further investigated in chrysanthemum using Agrobaterium-mediated genetic transformation, their downstream genes will be identified via RNA-seq. Taken together, the mechanism of CmTPL in regulating the flowering time will be revealed through the present proposal, and the function of SL in flowering time regulation will be enriched, which will provide excellent flowering genes for molecular breeding of chrysanthemum.

菊花具有很高的观赏和经济价值，在花卉产业中具有重要的地位，其周年供应需要光温调控，增加了生产成本。实验室已经发现独脚金内酯（SL）合成路径的关键基因CmCCD8超表达转基因菊花开花提前，对超表达转基因菊花转录组测序，发现SL信号路径基因CmTPL表达下调，开花相关的关键基因显著上调，CmTPL超表达拟南芥开花推迟。本研究拟在酵母双杂交筛选CmTPL互作蛋白CmJ的基础上，对筛选得到的蛋白进行体内体外互作验证，并利用转基因菊花分析CmTPL和CmJ功能，转录组测序筛选其下游路径基因，从而揭示CmTPL在菊花花芽分化过程中的作用机制。该项目有望在SL信号调控花期的分子机制方面取得突破，为菊花开花的分子育种提供优异的基因。

菊花（Chrysanthemum morifolium）是我国十大传统名花和世界四大切花之一，具有极高的观赏和经济价值，在花卉生产和园林产业应用中占至关重要的地位。为实现其周年供应，需要进行光温调控，但生产成本高昂。本研究以传统菊花‘神马’为材料，克隆了菊花‘神马’CmTPL1-2，组织定量发现CmTPL1-2基因在茎尖中的表达量最高，在根和茎中表达量很低。通过洋葱表皮亚细胞定位和转录活性验证实验表明CmTPL1-2定位在细胞核且具有转录抑制性。将CmTPL1-2、mut-CmTPL1-2转化拟南芥，发现CmTPL1-2超表达株系开花推迟，而mut-CmTPL1-2超表达株系则开花提前。通过对关键开花基因FT、TSF、FUL和AP1进行定量验证发现其相对表达量在CmTPL1-2超表达株系中下调，而在mut-CmTPL1-2超表达株系中上调。通过对CmTPL1-2转基因菊花进行转录组测序，发现光周期路径相关基因除TOC1/ PRR1之外，包括CO、COLs、CCA1、LHY、PRR7、AP2/ TOEs均下调表达，赤霉素（GA）路径相关基因GRAS/DELLAs、GA2ox和温敏路径基因SVP均显著上调，相应的下游关键开花调控基因FT、AGL20、AGL42、AP1/FUI和FUL/CDM41等表达下调。据此推测CmTPL1-2在转录水平通过光周期、赤霉素和温敏路径调控菊花开花。以CmTPL1-2为诱饵蛋白筛选其互作蛋白，发现其与CmJ3和CmSVP互作。以洋葱BiFC和烟草FLuCI蛋白互作验证手段证实了CmTPL1-2、CmSVP和CmJ3之间存在两两互作关系。在拟南芥中分别超表达CmSVP和CmJ3发现，CmSVP转基因株系开花推迟，CmJ3转基因株系开花提前。通过遗传分析表明，CmSVP-CmTPL1-2转录复合物是SVP作为花抑制因子的先决条件，以此来降低CmFTL3的转录活性。另外，CmSVP具有恢复拟南芥svp-31突变体的表型，并且AtSVP或CmSVP在拟南芥显性负突变tpl-1中的超表达对开花时间没有影响，说明SVP在两个物种中的功能高度保守。总之，我们揭示了SVP依赖TPL通过温敏开花途径抑制开花的机制，这将为菊花的分子育种提供优异的开花基因。发表SCI论文6篇；培养研究生4名。