光周期和糖信号诱导新铁炮百合成花的调控机理研究

Lily is one of the most important commercial flowers. Lilium× formolongi can reach flowering from seed within one year. The results of previous research showed that, the flowering inducement of L.× formolongi was closely related with photoperiod and carbohydrates. In this study, experiments of long day and short day treatments, reciprocal transfer and external application of sucrose are conducted to seedlings in different ages. Combining with flower bud differentiation process, temporal and spatial variations of carbohydrate content and expression patterns of CO，TPS1，FT genes to determine the juvenile, flowering competent, flowering induced, floral development stage and corresponding morphological parameter. After collecting the leaf and meristem samples of different stages, transcriptome and miRNA sequencing are carried on in order to predict the candidate genes and miRNA related to early flowering through bioinformatics and expression index analysis. The expressions of the selected candidate gene and miRNA are tested for various tissues with different development stages under different treatments, as well as conduct their function research through over expression vector construction. Finally, the pathway would be built based on molecular regulation mechanism information of photoperiod, sugar signals and miRNA on flowering of L.× formolongi. This study has important theoretical significance and practical application value not only on revealing the molecular regulation mechanism of early flowering of L.× formolongi, but also on breeding new cultivars with short growth period.

百合是重要的商品花卉，新铁炮百合可在一年内实现从播种育苗到开花结实。前期研究表明，其成花诱导与光周期密切相关。对不同苗龄的幼苗进行长短日照处理、交互迁光等处理，结合花芽分化、以及光周期途径中关键基因CO和FT基因的表达模式，确定幼龄期、感受态、开花决定和花发育的关键时间节点及形态指标。采集各时间节点的叶片和茎尖样本进行转录组测序，经生物信息学分析和不同样本间表达量的变化，找出与光周期成花调控相关的基因，预测可能与早花相关的候选基因。分析候选基因在不同组织、发育阶段和诱导条件下的表达模式，构建出新铁炮百合光周期调控成花的信号通路；克隆与早花相关的关键基因的全长，并进行序列分析。该研究对揭示新铁炮百合早花的分子机理及培育短生育期百合新品种具有重要理论意义和实际应用价值。

新铁炮百合实生苗可在一年内完成开花结实，光周期途径是其重要的开花途径之一。因此对不同苗龄的实生苗进行长短日照处理，通过形态、解剖、生理和基因表达等指标，确定诱导成花的关键节点。在转录组测序的基础上，获得新铁炮百合成花诱导途径的同源基因；研究光周期成花调控通路中重要同源基因在不同条件和发育阶段的表达模式，筛选出成花转变的关键基因。克隆获得基因全长，并进行序列分析。重要研究结果如下：. 在长日照条件下，新铁炮百合实生苗的开花时间较短日照下提前4个月左右。确定了新铁炮百合幼龄期、感受态、开花决定以及花发育的关键时间节点及相关的形态特征，其中幼龄期为播种后至7-8片基生叶，感受态期为9-10片基生叶，诱导期为抽葶并具1-2个节间，成年期为5-6个节间，花芽分化期为9-10个节间；在茎尖分生组织结构没有变化时，LfCOL9 和LfFT1的昼夜节律表达模式可作为新铁炮百合实生苗进入感受态和成花诱导期的重要指标。幼龄期、诱导期、成年期和分化期叶片和茎尖转录组测序共获得37,032个差异表达基因，共发现与成花相关的同源基因85个，其中转录因子52个，包括bZIP、RAV、MYB、CO-like、AP2/EREBP、SBP、GRAS、MADS-box等；这些基因分布于目前已发现的高等植物成花调控的五条途径，其中光周期成花调控差异表达基因41个。. 分析了光周期途径8个COLs基因家族同源基因以及LfFT1基因在不同光周期诱导条件下、不同发育阶段昼夜节律的表达情况。结果表明，LfCOL9和LfFT1基因在长日照下成花诱导时高表达，呈现出与拟南芥较为一致的昼夜节律表达模式；确定其是长日照诱导新铁炮成花转变的关键基因。从新铁炮百合中，克隆得到LfCOL5、LfCOL9、LfFT1、 LfLFY和LfAP1基因全长，经氨基酸序列系统进化分析，其与单子叶植物的同源基因亲缘关系较近。. 本研究首次获得百合光周期成花调控有关的基因数据库，对新铁炮百合早花机理的研究具有重要的理论意义。所获的开花关键基因，对培养生育期短的百合新品种及栽培技术的建立具有重要的实际应用价值。