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| Prokaryotic Expression, Polyclonal Antibody Preparation and Cytological Analysis of ZYP1 in Arabidopsis thaliana |
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Abstract To analyze the cytological behavior of synaptonemal complex (SC) related protein ZYP1 in plant meiosis, a recombinant vector pET28a-ZYP1 by cloning fragment of ZYP1 gene was constructed. The induction condition of the fusion protein expression in Escherichia coli was optimized. Polyclonal antibody was produced by immuning New Zealand rabbits (Oryctolagus cuniculus) with the protein which was purified by Ni-NTA affinity chromatography. The specificity and titer of the purified polyclonal antibody was examined by Western blot and ELISA. The results showed that the Arabidopsis thaliana ZYP1 prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 1.0 mmol/L isopropy-β-D-thiogalactoside (IPTG) at 37 ℃ for 4 h. The polyclonal antibody had high specificity and sensitivity. It was able to detect and locate the antegin of ZYP1 protein in plant cells by immunofluorescence staining. The results of immunofluorescence staining showed that ZYP1 protein appeared at leptotene and gradually reduced at diplonema and nearly disappeared at diakinesis. The ZYP1 protein was an important part of regulating the synapsis of homologous chromosomes in meiosis, and the dynamic changes was consistent with the formation and dissolution of the synaptonemal complex. This study is helpful to deep analysis of the biological functions of ZYP1 in plant meiosis, which is the related protein of synaptonemal complex.
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Received: 09 April 2015
Published: 11 October 2015
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