矮牵牛紫色花呈色关键基因筛选与PhWRKY44功能分析

Screening of key genes regulating purple pigmentation in petunia petals and functional analysis of PhWRKY44

摘要:

本研究旨在初步解析矮牵牛紫色花瓣呈色的分子调控机制，并挖掘关键调控基因。基于矮牵牛(Petunia×hybrida cv. Dreams Midnight)紫色花瓣3个发育阶段(S1浅绿、S2淡紫、S3深紫)的转录组分析，共鉴定出11 510个差异表达基因[错误发现率(false discovery rate, FDR)<0.05]。花青素合成途径关键基因PhANS、PhCHS、PhF3H、PhUFGT在S1-S3阶段显著上调，病毒诱导的基因沉默(virus-induced gene silencing, VIGS)实验证实其功能缺失导致花瓣紫色显著减退。转录因子筛选发现PhWRKY44表达量在S2和S3阶段的分别为S1阶段的4.96和5.13倍，PhMYB44、PhAN1及PhBHLH48亦呈现发育阶段特异性表达。本研究克隆了PhWRKY44，该基因全长1 482 bp，系统发育分析与保守结构域比对证实其为拟南芥WRKY44同源基因，编码54.93 kDa核定位碱性蛋白。该基因在花器官表达量最高，为根系的2.36倍，沉默后的表达量降至对照的64%，花青素含量降低45%，花瓣呈色显著减弱。本研究揭示了PhWRKY44通过正调控花青素合成影响花色形成，为观赏植物分子育种提供了新靶点。

Abstract:

This study aims to preliminarily elucidate the molecular regulatory mechanism underlying purple pigmentation in petunia petals and identify key regulatory genes. Based on transcriptomic analysis of Petunia×hybrida ‘Dreams Midnight’ at three developmental stages (S1: light green, S2: pale purple, S3: deep purple) of purple pigmentation in petals, we identified 11 510 differentially expressed genes (FDR<0.05). Key anthocyanin biosynthetic pathway genes PhANS, PhCHS, PhF3H, and PhUFGT were significantly upregulated during S1-S3. Virus-induced gene silencing confirmed that their functional loss caused significant fading of petal purple coloration. Transcription factor screening revealed that PhWRKY44 expression at S2 and S3 reached 4.96 folds and 5.13 folds, respectively, of that at S1, and PhMYB44, PhAN1, and PhBHLH48 also exhibited stage-specific expression patterns. PhWRKY44 was cloned, with a full length of 1 482 bp. Phylogenetic analysis and conserved domain alignment confirmed it as an ortholog of Arabidopsis WRKY44, encoding a nuclear-localized alkaline protein of 54.93 kDa. This gene showed the highest expression in floral organs, which reached 2.36 folds of root levels. Its silencing reduced PhWRKY44 expression to 64% of the control, decreased the anthocyanin content by 45%, and significantly weakened petal coloration. This study demonstrates that PhWRKY44 influences flower color formation by positively regulating anthocyanin biosynthesis, providing a new target for molecular breeding in ornamental plants.