关键词: 苹果, 转录因子, HD-Zip, MdHB1, 原核表达, 多克隆抗体

Abstract: HD-Zip transcription factors play an important role in plant growth and development, and studies at the protein level contribute to further elucidating their functions. Herein, the MdHB1 transcription factor gene from apple (Malus domestica) was cloned into the expression vectors pGEX-6p-1 and pET-28a(+), which were then transferred into BL21 (DE3) competent cells by heat shock for expression using isopropyl-β-D-thiogalactoside (IPTG) as an inducer. MdHB1-GST and MdHB1-His6 fusion proteins were obtained. SDS-PAGE analysis showed that at 18, 28 and 37 ℃, MdHB1-GST fusion protein existed mainly in the form of inclusion body and at lower temperatures (18 and 28 ℃) the ratio of soluble fusion proteins to the total proteins was increased. At 28 ℃, the content of fusion protein induced by 50 mg/L IPTG reached its maximum value after about 8 h, irrespective of different IPTG concentrations (10, 50 and 100 mg/L). Polyclonal antibody was prepared by immunizing adult rabbits with the MdHB1-His6 fusion protein purified by Ni-NTA column chromatography. Enzyme-linked immunosorbent assay (ELISA) showed that the polyclonal antibody had a high titer. Western blot assays showed that the polyclonal antibody could specifically recognize the MdHB1 protein in Escherichia coli and young apple leaves. Taken together, the polyclonal antibody could be used to explore the function of MdHB1 in plants.

Key words: Malus domestica, transcription factors, HD-Zip, MdHB1, prokaryotic expression, polyclonal antibody