金鱼草AmPIF4基因克隆及调控花香物质合成释放功能分析

Isolation and Characterization of AmPIF4 Gene in the Regulating Floral Scent Synthesis and Release in Snapdragon

为了研究光敏因素互作因子(phytochrome-interacting factors,PIFs)在光诱导花香物质合成释放中的调控作用,该研究从‘马里兰’金鱼草(Antirrhinum majus‘Maryland True Pink’)中克隆PIF4转录因子(AmPIF4)编码区序列,进行生物信息学分析、亚细胞定位和瞬时表达载体转化烟草,研究AmPIF4蛋白在细胞中的分布;并采用qRT-PCR技术检测AmPIF4基因的时空表达,运用病毒诱导的基因沉默(VIGS)验证基因功能,采用ATD-GC/MS技术测定花香成分含量。结果显示:(1)成功克隆到AmPIF4基因开放阅读框全长1497 bp,编码498个氨基酸;理论分子量55.58 kD,理论等电点6.44;同源比对分析显示AmPIF4蛋白与芝麻(Sesamum indicum)序列相似度最高。(2)AmPIF4蛋白主要定位于细胞核。(3)实时定量分析表明,AmPIF4基因在金鱼草各个时期及不同器官、组织中均有表达,而在花盛开期达到最高,其中在盛开期的叶片中表达量最高,但叶片与茎和萼片中的表达差异不显著。(4)基因沉默使金鱼草花瓣中AmPIF4基因的相对表达量较野生型显著下调约65%,且与阴性对照相比AmPIF4表达量也显著降低,同时使金鱼草花瓣中主要花香挥发物成分月桂烯、罗勒烯、苯甲酸甲酯较野生型分别显著增加了22%、24%、12%,花香释放量总体含量上升。研究表明,AmPIF4对花香物质合成释放有负调控作用,可能参与光对花香次生代谢的调控。

In order to explore the regulatory role of phytochrome-interacting factors(PIFs)in light induced floral scent synthesis and release,we cloned the coding region sequence from Antirrhinum majus‘Maryland True Pink’,bioinformatic analysis AmPIF4 were performed,and the subcellular location transient expression vector was used to transform tobacco and study the distribution of AmPIF4 protein in the cells in this study,and the temporal expression of AmPIF4 gene was detected by qRT-PCR.The function of AmPIF4 was verified via virus-induced gene silencing(VIGS),and the content of floral scent component was determined by ATD-GC/MS.The results showed that:(1)the ORF(open reading frame)of AmPIF4 gene was successfully cloned.It had a full-length of 1497bp,encoded 498 amino acids,and the theoretical isoelectric point and molecular weight of the encoding protein was 6.44 and 55.58 kD,respectively.Homology analysis showed that AmPIF4 protein had the highest sequence similarity to Sesamum indicum.(2)Subcellular localization assay showed that AmPIF4 protein was specially expressed in the nucleus.(3)qRT-PCR analysis showed that AmPIF4 gene was expressed in the whole development period and different organs,and reached the highest level in fully-opened period,among them,the highest expression level was found in the leaves of the fully-opened period,but the difference of the relative expression in the leaves,stems and sepals was not significant.(4)The silence of AmPIF4 gene significantly reduced the relative expression of AmPIF4 gene by about 65%compared with wild-type in snapdragon petals,it was also remarkably reduced compared with the negative control.At the same time,the main floral volatile components in snapdragon petals,myrcene,ocimene,and methyl benzoate,were significantly increased by 22%,24%,and 12%respectively compared with the wild type,the overall content of floral releases increased.This study suggests that AmPIF4 plays as a negative regulator in floral scent synthesis and release,and may participate in the regulation of light induced floral fragrance biosynthesis.