LfCOLs调控新铁炮百合早花的功能解析

Lilium × formolongi is an important bulb flower which can blossom in one year from seeds. Based on our researches, the photoperiod is the very important environmental signal for its flowering. CONSTANS (CO) plays a key role in the photoperiod pathway. The previous results indicated there were 8 members in LfCOLs family, and at least two genes could be related to the flowering induction in Lilium × formolongi. So, all LfCOLs members and other upstream and downstream genes of LfCOLs will be cloned, the characterization and bioinformatics analysis will be carried out. The temporal and spatial expression patterns of LfCOLs plus its upstream and downstream genes will be tested under long days and short days and reciprocal transfer photoperiod, the members which related to flowering regulation will be selected out. The interaction between LfCO and LfFKF1 proteins as well as LfCO protein and LfFT1 promoter will be analyzed through yeast two hybrid and single hybrid technology. After constructing the over expression and RNAi vectors of candidate genes, transformation will be carried out in Arabidopsis and Lilium × formolongi. Through that, the expression patterns of LfCOLs and its upstream/downstream genes and the flowering performance comparisons will be analyzed between the control and transgenic plants under the long day and short day conditions. Based on the above research, the flowering regulation mechanism of LfCOLs in photoperiod pathway will be determined for Lilium × formolongi. This research will elucidate the flowering induction of photoperiod pathways in Lilium, which have not only important theoretical meaning for flowering regulation mechanism study in lily, but also significant application value in lily’s flowering time regulation.

新铁炮百合是重要的球根花卉，实生苗可在一年内开花；前期研究结果显示，光周期是调控开花的重要环境信号，该途径核心因子CONSTANS（CO）在新铁炮百合中有8个成员，其中至少有2个成员参与实生苗的成花诱导，且分属两个类型，与拟南芥和水稻中的CO结构不同。因此，克隆新铁炮百合中LfCOLs家族成员及上下游基因并进行生物信息学分析，对长短日照及交互迁光条件下这些基因和花分生组织决定基因的表达模式进行比较分析，筛选出调控开花的候选LfCOLs。利用酵母双杂和单杂，解析LfCOLs蛋白与LfFKF1蛋白互作及LfCOLs蛋白与下游LfFT1的互作。构建候选基因过表达和RNAi载体，转化拟南芥和新铁炮百合，比较对照和转基因植株在长短日照下的开花性状和LfCOLs及上下游基因的表达，确定LfCOLs调控新铁炮百合早花的作用机理。本项目对阐释百合成花机理和花期改良分子育种具有重要的理论意义和实际应用价值。

新铁炮百合实生苗可在一年内开花，光周期是调控其开花的主要途径。为解析新铁炮百合光周期途径中CONSTANS（CO）基因的功能和作用机制，克隆了8个LfCOL基因家族成员及其上下游关键基因，并对不同光周期条件下的时空表达模式和昼夜节律进行了系统研究，得出LfCOL5、LfCOL6、LfCOL9和LfFT1在长日照条件下成花诱导期高表达，且呈现有规律的昼夜振荡表达模式，表明这三个基因具有促进开花的功能。LfCOL基因的下游基因为FT基因，首次在新铁炮百合中克隆并鉴定得到3个LfFT基因家族成员，经新铁炮百合及不同成花途径的百合品种中LfFTs基因时空表达模式分析以及拟南芥转化植株的功能验证，确定LfFT1在新铁炮百合成花诱导过程中发挥重要促进作用，LfFT3参与低温春化途径的成花调控，LfFT2与新铁炮百合鳞茎发生和发育相关。光周期和温度共同影响新铁炮百合实生苗植物的形态和开花，高温抑制实生苗的抽葶和开花，维持营养生长，而低温会逆转这种抑制。通过不同条件下转录组测序结果分析得出：昼夜节律钟和光周期成花途径关键基因以及成花整合因子均在高温时受到抑制， LfFKF1， LfCOLs等基因的表达模式受到干扰。利用酵母双杂交技术对LfCOL蛋白间及LfCOL蛋白与核心模块中的蛋白互作进行了分析，结果显示，成花促进基因中，LfCOL5蛋白与大部分LfCOLs蛋白、LfFKF1、LfGI和LfSPA1蛋白有明显互作；成花抑制因子中，LfCOL15、LfCOL13和LfCOL16的蛋白互作特性相似，分别与LfFKF1、LfGI蛋白发生互作，晚花基因存在功能冗余现象。分别构建了8个LfCOL基因的植物表达载体转化拟南芥co晚花突变体或野生型，其中LfCOL5、LfCOL6和LfCOL9能够显著促进拟南芥突变体开花，且比野生型早6-7天开花，是新铁炮百合重要的成花促进基因；而LfCOL15、LfCOL13和LfCOL16起到抑制开花的作用，尤其是转LfCOL15株系，无论在长短日照条件下都生长缓慢、开花晚，AtFT基因的表达都受到抑制。本项目研究结果对解析新铁炮百合光周期调控的成花机理和花期改良具有重要的理论意义和实际应用价值。