石榴花发育相关基因PgSPL2的克隆及功能研究

Cloning and Functional Study of the PgSPL2 Gene Related to the Development of Pomegranate Flowers

探究石榴SPL家族基因PgSPL2(XP_031375027.1)在石榴花发育中的作用,为进一步研究石榴花发育的机制奠定基础。以‘突尼斯’软籽石榴为试材,采用同源克隆技术克隆SPL家族基因PgSPL2,分析该基因的理化性质,以及在石榴不同组织中的表达差异,利用RT-q PCR分析生长调节剂处理时该基因的时空表达特性,转化拟南芥,验证该基因在花发育中的作用。结果表明,该基因包含一个长为591 bp开放阅读框,编码196个氨基酸,多重序列比对发现,该基因含有一个保守的SBP结构域和核定位信号,属于SBP转录因子基因家族;系统进化树分析表明,PgSPL2与拟南芥AtSPL3、AtSPL4和AtSPL5聚为一簇。RTqPCR结果表明,PgSPL2在果实和花蕾中的表达量相对较高,且生长调节剂喷施处理后,该基因在完全花中表达显著下调,同时,石榴完全花比率同步显著提高,初花、盛花期延迟。过量表达PgSPL2拟南芥出现早花现象,花序下游调控基因表达量显著提高。PgSPL2在石榴花发育中起调控开花时间的功能。

This study is aimed to investigate the role of the pomegranate(Punica granatum L.)SPL family gene PgSPL2(XP_031375027.1)in pomegranate flower development and lay a foundation for further research on the mechanism of pomegranate flower development. Using ‘Tunisian’ soft-seeded pomegranate as experimental material,we used homologous cloning technique to clone the SPL family gene PgSPL2,analyze the physicochemical properties of the gene and the expression differences in different tissues of pomegranate.Then we applied RT-qPCR to analyze the spatio-temporal expression characteristics of the gene upon growth regulator treatment. Finally we transformed Arabidopsis and verified the role of the gene in flower development. The results showed that the gene contained a 591 bp open reading frame encoding 196 amino acids,and multiple sequence alignment revealed that the gene contained a conserved SBP structural domain and nuclear localization signal,belonged to the SBP transcription factor gene family. Phylogenetic tree analysis showed that PgSPL2 clustered with Arabidopsis AtSPL3,AtSPL4 and AtSPL5. The RT-qPCR results showed that the expression of PgSPL2 was relatively high in fruits and flower buds,and the expression of PgSPL2 in complete flowers was significantly down-regulated after spraying with growth regulator,while the ratio of complete flowers significantly increased simultaneously,and the first flowering and blooming periods delayed. Overexpression of PgSPL2 in Arabidopsis presented early flowering and significantly higher expression of inflorescence downstream regulatory genes. These results indicate that PgSPL2 plays a role in regulating flowering time in pomegranate flower development.