title: Molecular cloning, polyclonal antibody preparation, and characterization of a functional iron-related transcription factor IRO2 from Malus xiaojinensis.
abstract: Transcription factors play important roles in plant growth and responses to environmental stresses. In this study, a novel basic helix-loop-helix iron-related transcription factor, IRO2, containing a 762-bp open reading frame and encoding 253 amino acids, was cloned from the iron-efficient genotype of Malus xiaojinensis. Localization analyses in onion showed that the MxIRO2 protein was targeted to the nucleus and activation studies in yeast indicated MxIRO2-BD had weak transcriptional activation activity. Prokaryotic expression of MxIRO2 in Escherichia coli BL21 (DE3) pLysS cells resulted in high expression levels of the protein when induced with isopropyl-β-d-thiogalactoside. The fusion protein was purified using Ni-NTA His-bind resin, and the purified MxIRO2-His fusion protein was used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was purified by precipitation with 50% saturated ammonium sulfate and DEAE Sephadex A-50 chromatography to obtain the immunoglobulin G fraction. The expression of MxIRO2 in roots and leaves of M. xiaojinensis seedlings under iron deficiency was determined. The results indicated that MxIRO2 was induced in both roots and leaves under iron deficiency. In these experimental conditions, the transcription and translation levels first increased and then decreased under iron deficiency. This work offers an important basis for further investigating the mechanisms of fruit tree adaptation to iron deficiency.
authors.affiliation: Institute for Horticultural Plants, China Agricultural University, Yuanmingyuan West Road, Haidian District, Beijing 100193, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Yin
authors.affiliation: Institute for Horticultural Plants, China Agricultural University, Yuanmingyuan West Road, Haidian District, Beijing 100193, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute for Horticultural Plants, China Agricultural University, Yuanmingyuan West Road, Haidian District, Beijing 100193, China.
authors.forename: Mudan
authors.initials: M
authors.lastname: Yan
authors.affiliation: Institute for Horticultural Plants, China Agricultural University, Yuanmingyuan West Road, Haidian District, Beijing 100193, China.
authors.forename: Xinzhong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute for Horticultural Plants, China Agricultural University, Yuanmingyuan West Road, Haidian District, Beijing 100193, China.
authors.forename: Haifa
authors.initials: H
authors.lastname: Pan
authors.affiliation: Institute for Horticultural Plants, China Agricultural University, Yuanmingyuan West Road, Haidian District, Beijing 100193, China.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Xu
authors.affiliation: Institute for Horticultural Plants, China Agricultural University, Yuanmingyuan West Road, Haidian District, Beijing 100193, China. Electronic address: rschan@cau.edu.cn.
authors.forename: Zhenhai
authors.initials: Z
authors.lastname: Han
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D027845:Malus / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; D010940:Plant Proteins; D014157:Transcription Factors
keywords: Antibody preparation; Expression; Iron deficiency; Prokaryotic expression; Purification