FaARF4调控草莓提早开花的分子机理研究

Flowering time of crops with fruit as the product will affect harvest period. It is of great significance to find the factors that determine the flowering time and to identify their regulatory mechanism. Our previous studies have found that FaARF4 transcription factor can regulate the flowering time of strawberry. The present research program will find out the target genes regulated by FaARF4 with transcriptome sequencing and qRT-PCR. The interaction between FaARF4 and its target genes will be confirmed by yeast one-hybrid system and electrophoretic mobility shift assay (EMSA). Its function in the flowering time regulation will be analyzed by adopting over-expression and silence of ARF4 target genes in strawberries, respectively. The subcellular localization of FaARF4 target protein will be investigated by transforming target gene::GFP into onion epidermal cells. According to GUS staining analysis, the promoter activity of ARF4 gene will be elucidated in the transgenic plants with proARF4::GUS which will be treated with different types and concentrations of hormones, etc. And the factors affecting the expression of ARF4 gene will be further confirmed. The aim of this project is to reveal the molecular mechanism of FaARF4 gene in regulating strawberry flowering. The findings will lay a foundation for flowering promotion or inhibition of strawberry by regulating the expression of ARF4 gene. The results will also provide theoretical reference for the research on flowering mechanism of other fruit trees.

以果实为产品器官的农作物开花早晚影响着产品收获期，弄清决定其开花早晚的因子并研究其调控机制具有重要意义。申请人前期研究发现，转录因子FaARF4具有调控草莓开花早晚的功能，因此本项目拟采用转录组测序和qRT-PCR技术，筛选FaARF4调控的下游靶基因，并借助酵母单杂交和EMSA试验，确定FaARF4与靶基因间的互作关系；通过FaARF4靶基因过量表达及沉默来分析其对草莓开花早晚的影响；利用FaARF4靶基因::GFP融合基因通过转化洋葱表皮细胞，进行ARF4靶蛋白的亚细胞定位；结合GUS组织染色分析转proARF4::GUS基因植株在不同激素种类、浓度等处理下启动子的转录活性，进而明确影响ARF4基因表达的因素。通过上述研究揭示FaARF4调控草莓开花的分子机理，为通过调控ARF4基因表达来提早或延晚草莓开花奠定基础，并可为其他果树作物开花机理研究提供理论参考。

开花早晚直接影响草莓生产的经济效益，研究草莓开花的分子机理具有重要的生产实践意义。本项目以草莓为试材，系统研究了ARF4基因调控草莓提早开花的分子机理。结果发现过表达ARF4基因提早森林草莓开花15 d，沉默ARF4基因延晚草莓开花8 d；qRT-PCR试验显示ARF4过表达草莓植株中AP1和FUL的表达量明显升高，沉默植株中AP1和FUL的表达量明显下降；FaARF4具有转录激活活性并且定位在细胞核上；Y1H、萤火虫荧光酶素报告基因试验、GUS报告基因试验证明了ARF4能够直接结合到AP1和FUL基因启动子上激活它们的表达；基因结构和功能分析显示FveAP1和FveFUL蛋白均定位在细胞核上，均为开花促进基因；ARF4基因的启动子活性分析显示IAA能够显著促进其表达，同时ARF4的表达也受上游miR390的间接负调控，既草莓中存在miR390-tasiRNA3-ARF4-AP1/FUL模块，它们的协同作用机制是miR390促进tasiRNA3的生物合成，而tasiRNA3负调控ARF4的表达，ARF4又通过促进AP1和FUL的表达来调控草莓提早开花。研究结果为通过调控ARF4基因表达来控制草莓开花时间奠定重要基础，为草莓早熟分子育种和其它果树作物开花机理研究提供参考。项目组完成了项目研究目标，培养1名博士研究生和3名硕士研究生，获得国家发明专利1项，发表论文6篇，其中SCI收录论文5篇。