牡丹PsAP3基因的克隆与表达分析

Cloning and expression analysis of PsAP3 gene in tree peony

优质牡丹花型育种是牡丹产业发展遭遇的瓶颈问题之一。本研究以牡丹(Paeonia sufruticosa)单瓣和雄蕊瓣化品种为植物材料,以调控牡丹雄蕊发育相关的关键基因PsAP3为切入点,通过基因克隆、亚细胞定位、表达分析初步解析了牡丹PsAP3基因的功能,筛选了PsAP3可能存在下游互作基因,构建了TRV2-PsAP3沉默载体并成功转化农杆菌,用于后续牡丹瞬时沉默试验。结果显示:牡丹PsAP3基因全长612 bp(GenBank登记号:MT225537),编码203个氨基酸,分子量为23891.18,属亲水蛋白,含有一个MADS结构域和一个K结构域。系统进化分析表明PsAP3与栓皮栎亲缘关系最近。亚细胞定位结果显示PsAP3蛋白定位在细胞核中。表达分析结果显示,与单瓣品种的雄蕊相比PsAP3在雄蕊瓣化品种的内瓣中表达量显著提高,这也预示着可能是PsAP3基因在在花器官中表达扩张,在雄蕊中显著高表达导致了牡丹的雄蕊瓣化现象。酵母双杂交结果表明PsAP3编码的蛋白与PsAP1-1、PsPI、PsSEP、PsAG编码的蛋白互作。本研究初步解析了牡丹PsAP3的功能,为牡丹雄蕊瓣化分子机理的揭示提供理论依据,并为进一步精细化调控牡丹花型奠定理论基础。

Superior flower type breeding is one of the bottleneck problems encountered by the tree peony(Paeonia suffruticosa)industry.In this study,the single flower and stamen petalody varieties were used as plant materials.As the entry point,the key gene PsAP3 regulating peony stamen development,was characterized by gene clone,subcellular localization,expression analysis and yeast two-hybrid.PsAP3 silencing vector was constructed and successfully transformed into Agrobacterium tumefaciens for subsequent transient silencing assays in peony.The results showed that the full length of PsAP3 gene is 612 bp(Gen-Bank accession number:MT225537),encoding 203 amino acids,with a molecular weight of 23891.18.It is a hydrophilic protein and contains a MADS domain and a K domain.Phylogenetic analysis showed that PsAP3 was most closely related to Quercus variabilis.Subcellular localization results showed that PsAP3 protein was localized in the nucleus.Expression analysis showed that PsAP3 expression was significantly higher in the inner petals of stamen petalized varieties compared to the stamens of single petal varieties.These indicated that the expansion of PsAP3 gene may be expanded in the floral tube and significant high expression in stamens might lead to stamen petalody in tree peony.Yeast two-hybrid results showed that PsAP3 protein interacted with PsAP1-1,PsPI,PsSEP and PsAG protein.This study provides a preliminary analysis of the function of PsAP3 in peony,provided a theoretical basis for revealing the molecular mechanism of stamen petalody,and laid a theoretical basis for further fine-controling the flower type in tree peony.