关键词: 香蕉, WRKY, 转录因子, 原核表达, 多克隆抗体, 耐冷性

Abstract: To investigate the function of the MaWRKY1 gene，a transcription factor previously cloned from banana fruit，the polyclonal antibody of MaWRKY1 was prepared. The recombinant plasmid pET-MaWRKY1 was constructed with 699 bp segments started from the 168 amino acid to the 400 amino acid including the two conserved motif WRKYGQK and an about 26 kD expressed His-MaWRKY1 fusion protein was identified by SDS-PAGE following the expression of the recombinant His-MaWRKY1 fusion protein in E.coli BL21（DE3）cells with high efficiency. About 0.5 mg • mL-1 fusion protein was obtained after puified by Ni-NTA agarosecolumn，separated by SDS-PAGE，excised from the gel and electroeluted，then the purified fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifth injection of antigen，the antiserum was obtained and further purified with method of immuno-precipitation. Indirect ELISA test and the western blot analysis showed that the obtained anti-MaWRKY1 polyclonal antibody have good sensitivity and high specificity with a titer of 1︰160 000. Furthermore，western blot assay revealed that a 26 kD peptide was specifically detected in the total proteins of different banana tissues with anti-MaWRKY1 polyclonal antibody，indicating that the antibody could react to the total proteins expressed in banana specifically. In addition，low temperature could induce the expression of MaWRKY1 protein in banana fruit，suggesting that the expression of MaWRKY1 protein in banana fruit was likely to be involved in regulating the chilling-tolerance of fruit.

Key words: banana, WRKY, transcription factor, prokaryotic expression, polyclonal antibody, chilling-tolerance