关键词: 苹果, 红肉, MdICE1, 花青苷, 酵母双杂交, 亚细胞定位, 原核表达

Abstract:

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 bHLH motif. Furthermore，low temperatures are conducive to anthocyanin biosynthesis in callus. The transcript levels of MdICE1 and anthocyanin biosynthesis related genes（MdMYB10 and MdbHLH3）were significantly higher in callus incubated at 8 ℃ than those at 24 ℃. The yeast two hybrid experiments showed that the MdICE1 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.

Key words: apple, red flesh, MdICE1, anthocyanin, yeast two hybrid experiment, subcellular localization assay, prokaryotic expression technique