油茶低磷响应转录因子CoPHR1、CoPHR2基因的克隆与表达分析

Cloning and expression analysis of low-phosphorus-responsive transcription factors CoPHR1 and CoPHR2 in Camellia oleifera

本研究以油茶(Camellia oleifera)‘华金’扦插苗根系为材料,通过RT-PCR克隆出MYB-CC转录因子家族中2个编码磷饥饿信号关键调控因子的基因,分别命名为CoPHR1和CoPHR2,通过生物信息学手段对其序列的理化性质、结构与功能进行了预测和分析。结果表明:2个基因编码序列(CDS)分别长879和939 bp,开放阅读框(ORF)分别为879和912bp,编码292和303个氨基酸。Blastx分析结果显示CoPHR1、CoPHR2与茶树(C. sinensis)、中国猕猴桃(Actinidia chinensis)的低磷响应转录因子(phosphate starvation response, PHR)氨基酸序列相似性最高,分别达88%和68%;其蛋白分子质量分别为31.74和33.50 kDa,理论等电点分别是6.39和6.46;两者均为不稳定的非分泌蛋白,二级结构则均以α螺旋和无规则卷曲为主。实时荧光定量PCR表明CoPHR1和CoPHR2在叶中的相对表达量均显著高于茎、根、根尖组织,且在老叶中受低磷诱导显著;亚细胞定位分析表明CoPHR1和CoPHR2所编码的蛋白质分别定位于细胞核与细胞膜。该研究结果拟为进一步了解油茶磷饥饿信号的调控途径提供重要依据。

Using the roots of Camellia oleifera ‘Huajin’ cuttings as materials, two key regulators of phosphorous starvation signals in the MYB-CC transcription factor family were identified by RT-PCR and they are named CoPHR1 and CoPHR2. The physicochemical properties, structure and function of CoPHR1 and CoPHR2 were predicted and analyzed by bioinformatics methods. The results showed that the cDNA of two genes were 879 and 939 bp in length and included an open reading frame(ORF) of 879 and 912 bp, encoding 292 and 303 amino acids respectively. Blastx analysis results showed that CoPHR1 and CoPHR2 had the highest amino acid sequence similarity to the phosphate starvation response(PHR) of C. sinensis and Actinidia chinensis, reaching 88% and 68% respectively;the molecular weights of CoPHR1 and CoPHR2 proteins were 31.74 and 33.50 kDa, respectively. The theoretical isoelectric point are 6.39 and 6.46, both of which are unstable non-secreted proteins;the secondary structures of both are dominated by are α-helix and irregular curl. Real-time quantitative PCR(qPCR) suggested that the relative expression levels of CoPHR1 and CoPHR2 in leaves were significantly higher than those in stem, roots, and root tips, and were induced by low phosphorus stress in old leaves significantly. Subcellular localization indicated that the proteins encoded by CoPHR1 and CoPHR2 were localized in the nucleus and cell membrane separately. The results of this study provide an important basis for an in-depth understanding of the regulation pathways of phosphorus-starvation signals in C. oleifera.