桂花OfbHLH89参与调控花开放的功能解析

Functional Analysis of OfbHLH89 Gene in Regulating Flower Opening in Osmanthus fragrans

在桂花(Osmanthus fragrans)转录组数据库中筛选并克隆出可能参与花开放过程的候选基因OfbHLH89。该基因的开放阅读框(ORF)长744 bp,编码247个氨基酸。氨基酸序列和系统进化树分析表明,OfbHLH89与红豆VaBPE和辣椒CbBPE的亲缘相似性分别为54.1%和52.74%。亚细胞定位显示OfbHLH89定位于细胞核。时空表达分析表明OfbHLH89在花中高表达,且在花开放过程中的表达水平逐渐升高,盛开期达到峰值;19℃低温诱导下的花芽中,OfbHLH89的转录水平显著高于23℃对照。OfbHLH89过表达矮牵牛花瓣变大,表明OfbHLH89可能参与花开放过程的花瓣扩张;检测转基因矮牵牛下游与细胞扩张相关基因的转录水平发现,PhEXPA1、PhXTH24和PhXTH25均显著上调。此外,在瞬时过表达OfbHLH89的桂花花芽中,OfXTH5、OfXTH21、Of XTH32、Of XTH34、OfEXPA4和OfEXPA13的相对表达量也显著高于对照花芽。综上,OfbHLH89可能通过激活细胞扩张相关基因的转录使花瓣变大从而影响桂花开放。

The flower opening process was investigated by screening and cloning gene OfbHLH89 from the Osmanthus fragrans transcriptome database.This gene has a coding frame(ORF)that is 744 bp long and encodes for 247 amino acids.Sequence alignment and phylogenetic tree analysis revealed that OfbHLH89 shared similarities of 54.1%and 52.74%with Vigna angularis VaBPE and Capsicum baccatum CbBPE,respectively.The subcellular localization results showed that OfbHLH89 was localized on the nucleus.Spatiotemporal expression analysis showed that OfbHLH89 was highly expressed in the flower,and its expression gradually increased from ball-shaped stage to full flowering stage,reaching a peak at the full flowering stage.Further analysis revealed that under low temperature induction,the relative expression level of OfbHLH89 in the flower buds was significantly higher than that of the control.In Petunia hybrida,overexpression of OfbHLH89 resulted in the enlargement of petals,suggesting that OfbHLH89 may be involved in petal expansion during flower opening in O.fragrans.Additionally,the expression of PhEXPA1,PhXTH24,and PhXTH25 were significantly up-regulated.Quantitative analysis of downstream cell expansion related genes by transient transformation of OfbHLH89 into O.fragrans flower buds revealed that the expression of OfXTH5,OfXTH21,OfXTH32,OfXTH34,OfEXPA4 and OfEXPA13 were significantly upregulated compared with empty vector.It is suggested that OfbHLH89 can impact the flower opening of O.fragrans by activating the transcription of genes related to cell expansion,leading to increase in petal size.