Purification, immunological, and functional characterization of MxFIT in Malus xiaojinensis

Abstract

MxFIT is a FER-like iron deficiency induced transcriptional factor in Malus xiaojinensis. Here, we described the heterologous expression of MxFIT in Escherichia coli BL21 (DE3) host cells. The E. coli harboring the recombinant construct pET-MxFIT was efficiently induced to express the MxFIT protein at a high level and the optimal profile for MxFIT expression was investigated. By inoculating a New Zealand rabbit with purified MxFIT-His fusion protein, a high specific anti-MxFIT antiserum was achieved. Western blot analysis showed MxFIT protein expression was induced in roots when iron supply was limiting and was inhibited when iron supply was excessive. In leaves, there was almost no expression irrespective of iron supply. The localization of MxFIT on transverse section of root indicated that MxFIT participated in iron deficiency response. Over-expression of MxFIT in transgenic tobacco suspension cells showed that MxFIT increased iron absorption under insufficient iron supply. This study provides a basis for further investigating the underlying mechanism of high iron absorption efficiency in M. xiaojinensis.