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

IF 0.8 4区 生物学 Q4 PLANT SCIENCES Biologia Plantarum Pub Date : 2021-06-11 DOI:10.32615/BP.2021.004
L. Yin, X. L. Chen, S. L. Ma, F. Li, F. Zhou, Yongyan Wang
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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.
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小金苹果MxFIT基因的纯化、免疫学及功能研究
MxFIT是小金苹果铁缺乏的转录因子。在这里,我们描述了MxFIT在大肠杆菌BL21 (DE3)宿主细胞中的异源表达。我们高效地诱导携带重组构建物pET-MxFIT的大肠杆菌高水平表达MxFIT蛋白,并研究了MxFIT的最佳表达谱。用纯化的MxFIT-His融合蛋白接种新西兰兔,获得了高特异性的抗mxfit抗血清。Western blot分析显示,当铁供应不足时,MxFIT蛋白在根中被诱导表达,当铁供应过剩时,MxFIT蛋白在根中被抑制表达。在叶片中,与铁供应无关,几乎没有表达。MxFIT在根横切面的定位表明MxFIT参与了缺铁反应。转基因烟草悬浮细胞中过表达MxFIT表明,在铁供应不足的情况下,MxFIT增加了铁的吸收。本研究为进一步探讨小金草高铁吸收效率的潜在机制提供了基础。
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来源期刊
Biologia Plantarum
Biologia Plantarum 生物-植物科学
CiteScore
2.80
自引率
0.00%
发文量
28
审稿时长
3.3 months
期刊介绍: BIOLOGIA PLANTARUM is an international journal for experimental botany. It publishes original scientific papers and brief communications, reviews on specialized topics, and book reviews in plant physiology, plant biochemistry and biophysics, physiological anatomy, ecophysiology, genetics, molecular biology, cell biology, evolution, and pathophysiology. All papers should contribute substantially to the current level of plant science and combine originality with a potential general interest. The journal focuses on model and crop plants, as well as on under-investigated species.
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