红肉苹果冷信号基因MdICE1的表达及其蛋白与MdMYB的互作

Expression of a Cold-induced Gene MdICE1 in Red Flesh Apple and Its Protein Interaction with MdMYB

根据拟南芥AtICE1蛋白序列在苹果基因组中Blast比对得到苹果同源蛋白序列,利用DNAMAN软件设计特异性引物并克隆苹果冷信号基因(MDP0000662999),暂命名为MdICE1。以从新疆红肉苹果与‘富士’杂交F_1代中选出的‘紫红3号’叶片诱导出的红色愈伤组织为试材克隆MdICE1,测序发现该基因的开放阅读框长度为1 626 bp,编码541个氨基酸。进化树分析表明,MdICE1与AtICE1在同一进化支上,推测它们具有相似的功能。氨基酸序列比对发现,MdICE1存在bHLH基序。低温处理有利于苹果愈伤组织花青苷的累积;与培养在24℃下的愈伤组织相比,低温(8℃)诱导苹果愈伤组织冷信号基因MdICE1以及花青苷合成相关转录因子基因MdMYB10和MdbHLH3的表达。酵母双杂交试验证明MdICE1可以与MdMYB10相互作用;亚细胞定位发现MdICE1蛋白存在于细胞核内;转化大肠杆菌并诱导获得了MdICE1的重组蛋白,为进一步研究MdICE1蛋白在花青苷代谢途径中的功能奠定了基础。

The red-callus induced from the leaves of‘Zihong 3＇apple in F1 population of Malus sieversii f. niedzwetzkyana crossed with‘Fuji＇was used as materials. A cold signal relative gene（MDP0000662999）was obtained from apple genome by BLAST searches of the Arabidopsis AtICE1 protein sequence,designated MdICE1. Sequence analysis indicated that the open reading frame（ORF）length of MdICE1 was 1 626 bp,which encoded 541 amino acids. Phylogenetic analysis revealed that MdICE1 was homologous with AtICE1 which was involved in the cold signaling. The aligned protein sequences also revealed that MdICE1 contains the b HLH motif. Furthermore,low temperatures areconducive to anthocyanin biosynthesis in callus. The transcript levels of MdICE1 and anthocyanin biosynthesis related gene（sMdMYB10 and MdbHLH3）were significantly higher in callus incubated at 8 ℃ than those at 24 ℃. The yeast two hybrid experiments showed that the Md ICE1 could interact with MdMYB10. The subcellular localization assays revealed that MdICE1 was located in the nucleus. In addition,the recombinant protein of MdICE1 was obtained by the prokaryotic expression technique,which laid a foundation for MdICE1 functional identification associated with anthocyanin metabolism.