A novel transcription factor FnMYB4 regulates pigments metabolism of yellow leaf mutants in Fragaria nilgerrensis

The strawberry species Fragaria nilgerrensis Schlechtendal ex J.Gay,renowned for its distinctive white,fragrant peach-like fruits and strong disease resistance,is an exceptional research material.In a previous study,an ethyl methane sulfonate(EMS)mutant library was established for this species,resulting in various yellow leaf mutants.Leaf yellowing materials are not only the ideal materials for basic studies on photosynthesis mechanism,chloroplast development,and molecular regulation of various pigments,but also have important utilization value in ornamental plants breeding.The present study focused on four distinct yellow leaf mutants:mottled yellow leaf(MO),yellow green leaf(YG),light green leaf(LG),and buddha light leaf(BU).The results revealed that the flavonoid content and carotenoid-to-chlorophyll ratio exhibited a significant increase among these mutants,while experiencing a significant decrease in chlorophyll and carotenoid contents compared to the wild type(WT).To clarify the regulatory mechanisms and network relationships underlying these mutants,the RNA-seq and weighted gene coexpression network(WGCNA)analyses were employed.The results showed flavonoid metabolism pathway was enriched both in MO and YG mutants,while the chlorophyll biosynthesis pathway and carotenoid degradation pathway were only enriched in MO and YG mutants,respectively.Subsequently,key structural genes and transcription factors were identified on metabolic pathways of three pigments through correlation analyses and quantitative experiments.Furthermore,a R2R3-MYB transcription factor,FnMYB4,was confirmed to be positively correlated with flavonoid synthesis through transient overexpression,virus-induced gene silencing(VIGS),and RNA interference(RNAi),accompanying by reoccurrence and attenuation of mutant phenotype.Finally,dual-luciferase(LUC)and yeast one-hybrid assays confirmed the binding of FnMYB4 to the FnFLS and FnF3H promoters,indicating that FnMYB4 positively regulates flavonoid synthesis.In addition,correlation analyses suggested that FnMYB4 also might be involved in chlorophyll and carotenoid metabolisms.These findings demonstrated the pivotal regulatory role of FnMYB4 in strawberry leaf coloration.

野生黄毛草莓NAC转录因子基因FnNAC7克隆与表达分析

NAC转录因子基因为植物所特有,在植物的生长发育、非生物和生物胁迫的抗逆反应中起着重要作用。本研究以野生黄毛草莓(Fragaria nilgerrensis Schidl.)为材料,利用RT-PCR技术,从中克隆到1个NAC转录因子基因FnNAC7(GenBank登录号:MK685675)。序列分析结果表明,FnNAC7开放阅读框为1137 bp,编码378个氨基酸,包含一个保守的NAM结构域。同源性分析结果表明,FnNAC7基因编码的氨基酸序列与森林草莓编码的氨基酸相似性为94.87%。蛋白结果预测显示,FnNAC7蛋白为不稳定的酸性疏水蛋白,不存在跨膜结构域,且无信号肽存在;亚细胞定位试验结果显示,该基因编码的蛋白质在细胞核中表达;实时定量PCR结果显示,接种草莓尖孢炭疽菌(Colletotrichum acutatum)后,草莓叶片FnNAC7基因表达上调,接种24 h后该基因表达量最高,是对照组的17.0倍。研究结果为进一步深入开展NAC基因在草莓炭疽病抗性中功能分析和分子育种提供了科学的依据,也为进一步研究FnNAC7基因在野生黄毛草莓抗草莓炭疽病中所起的调控作用提供了参考。